Fixaxfx bispecific antibody with common light chain

ABSTRACT

Bispecific antigen binding molecules (e.g., antibodies) that bind blood clotting factors, factor IXa (FIXa) and factor X (FX), and enhance the FIXa-catalysed activation of FX to FXa. Use of the bispecific antigen binding molecules to control bleeding, by replacing natural cofactor FVIIIa which is deficient in patients with haemophilia A.

FIELD OF THE INVENTION

This invention relates to bispecific antigen-binding molecules (e.g., antibodies) that bind factor IXa and factor X clotting factors in the blood coagulation cascade. Such bispecifics functionally substitute for factor VIII by activating factor X, restoring blood clotting ability to patients who are deficient in FVIII, i.e., patients who have type A haemophilia.

BACKGROUND

Haemophilia is an inherited condition in which the blood has a reduced ability to clot, owing to loss of function (partial or total) of one of the many clotting factors. Haemophilia A is a deficiency in blood clotting factor VIII (FVIII). The disease has mild, moderate and severe forms, depending on the degree to which the patient retains any residual FVIII function and on the balance of other components in the blood coagulation cascade. If untreated, haemophilia A leads to uncontrolled bleeding, which can result in severe disability, especially through damage to joints from haemarthrosis events. The disease is often life-limiting and can be life-threatening. The global incidence of haemophilia A is believed to be around 1:10,000. Haemophilia B (deficiency of a different blood clotting factor, factor IX) is less common, with an incidence of around 1:50,000. Both diseases are X-linked so are usually found in males, the incidence of haemophilia A in male births thus being around 1 in 5,000.

Preventing bleeding episodes is essential to improving patients' quality of life and reducing the risk of fatal blood loss. For haemophilia A, the missing co-factor can be replaced by administration of FVIII. FVIII for administration to a patient may be recombinantly expressed or it may be purified from blood plasma. Typically, patients on this treatment self-inject with FVIII every 48 hours or 3× per week.

Treatment with FVIII is not a perfect solution. A serious drawback is that it can trigger production of allo-antibodies in the body. This renders treatment with FVIII ineffective, as the allo-antibodies bind the FVIII and prevent its activity, putting the patient in a dangerous situation if a bleed occurs. Such inhibitory antibodies develop in about 30% of patients treated with FVIII for severe haemophilia.

Treatment with plasma-derived FVIII, rather than the recombinant form, has been reported to have a lower risk of triggering inhibitory antibodies in patients. This may be due to the plasma-derived form retaining Von Willebrand factor (VWF), which is found naturally in association with FVIII and may mask immunogenic epitopes. However, no form of FVIII has yet been produced that completely avoids the risk of inhibitory antibodies.

Despite being possibly more immunogenic, recombinant FVIII offers some advantages over the plasma-derived form, since being more stable it is easier and cheaper to store and transport. The risk of transmitting infections via products from donated blood plasma is now much reduced compared with the 1980s when viruses such as hepatitis C and HIV were inadvertently spread to recipients of infected blood products, but of course the need for strict safety controls remains.

New recombinant forms of FVIII have been developed, such as the B-domain truncated polypeptide turoctocog alfa (NovoEight®). However, such products are ineffective for patients that develop neutralising antibodies against FVIII. Some patients successfully undergo immune tolerance induction to prevent anti-FVIII antibodies from developing. However, there remains a substantial demand for alternatives to FVIII for use in patients who have, or are at risk of developing, inhibitory antibodies.

One such alternative is recombinant factor Vila, known as activated eptacog alfa (NovoSeven®). However, it has a short half-life and must be injected every few hours. Its use is largely restricted to rescue therapy or providing haemostatic cover during surgery in haemophiliacs who have inhibitory antibodies, rather than being a viable option for long term protective treatment.

Another available product is FEIBA (Factor Eight Inhibitor Bypassing Activity), an activated prothrombin complex concentrate (aPCC), which similarly can be used to control bleeding episodes and to prevent bleeding during surgical interventions in haemophiliac patients who have inhibitors to factor VIII.

A variety of other alternative therapies are currently being pursued, such as gene therapy, suppression of anti-thrombin using siRNA, and an antibody to TFPI (Tissue factor Pathway Inhibitor), concizumab.

One approach is a humanised bispecific IgG antibody targeting both factor IXa (FIXa) and factor X (FX). The bispecific antibody binds FIXa with one arm and FX with the other arm, bringing these two co-factors together and thereby promoting FIXa-catalysed activation of FX in the same way that FVIII does. Thus, the antibody functionally replaces FVIII in the blood coagulation cascade (FIG. 1). As its structure is completely different from FVIII, the antibody cannot be neutralised by anti-FVIII antibodies and so is suitable for patients who have developed, or are at risk of developing, allo-antibodies to administered FVIII.

In 2012, Kitazawa et al reported isolation of a FIXa/X bispecific antibody which was able to activate FX, from a screen of approximately 40,000 anti-FIXa/X bispecific antibodies that had been produced by immunising 92 laboratory animals with human FIXa or FX and co-transfecting the anti-FIXa and anti-FX antibody genes into host cells for expression [1]. The selected antibody was refined to generate a humanised antibody designated hBS23, which showed coagulation activity in FVIII-deficient plasma and in vivo haemostatic activity in primates [1]. A more potent version of this antibody, designated hBS910 [2], entered clinical trials under the investigational drug name ACE910, INN emicizumab [3]. The development of ACE910 took place in one of the leading antibody groups globally. Nevertheless, it took more than 7 years to engineer a molecule with the appropriate in vivo efficacy and with biochemical and biophysical properties suitable for clinical scale manufacturing.

In a phase I study of 48 healthy male subjects receiving ACE910 subcutaneously at doses up to 1 mg/kg, 2 subjects tested positive for anti-ACE910 antibodies [4]. The antibody was reported to have a linear pharmacokinetic profile and a half-life of about 4-5 weeks [4]. Emicizumab was subsequently administered to 18 Japanese patients with severe haemophilia A, at weekly subcutaneous doses of up to 3 mg/kg, and was reported to reduce the episodic use of clotting factors to control bleeding in these patients [5]. In December 2016, emicizumab was reported to have met its primary endpoint in a phase III clinical trial for reducing bleeding in patients with haemophilia A (the “HAVEN 1” study). A statistically significant reduction in the number of bleeds was reported for patients treated with emicizumab prophylaxis compared with those receiving no prophylactic treatment. The study was also reported to have met all secondary endpoints, including a statistically significant reduction in the number of bleeds over time with emicizumab prophylaxis treatment in an intra-patient comparison in people who had received prior bypassing agent prophylaxis treatment. The efficacy data on emicizumab are therefore encouraging, although safety concerns were heightened by the death of a patient on the HAVEN 1 study. The approved drug carries a boxed warning regarding the risk of thrombotic microangiopathy and thromboembolism in patients receiving aPCC in combination with emicizumab. As noted above, aPCC is used to control bleeding in patients who have inhibitory antibodies to FVIII, a key patient group for treatment with the bispecific antibody.

It is important to note that management of haemophilia requires continuous treatment for a patient's lifetime, beginning at the point of diagnosis—which is usually in infancy—and calls for a therapy that will be tolerated without adverse effects and that will remain effective over several decades or even a century. Long term safety, including low immunogenicity, is therefore of greater significance for an anti-haemophilia antibody compared with antibodies that are intended to be administered over a shorter duration such as a period of weeks, months or even a few years.

WO2018/098363 described bispecific antibodies binding to FIX and FX, isolated from a human antibody yeast library (Adimab). WO2018/098363 disclosed that increasing the affinity of the anti-FIXa arm of a bispecific antibody results in an increase in FVIIIa activity (represented by decreased blood clotting time in an assay). A bispecific antibody “BS-027125” was generated by affinity maturation of an initially selected “parent” antibody, which increased the affinity of its FIXa-binding arm. BS-027125 was reported to achieve approximately 90% FVIIIa-like activity in a one-stage clotting assay. When compared with emicizumab, BS-027125 was reported to exhibit much higher affinity binding to factor FIX zymogen, FIXa and FX zymogen, and much lower binding (no detected binding) to FXa. The FIX-binding arm, “BIIB-9-1336” reportedly showed selective binding for FIXa (activated FIX) in preference to FIX zymogen (mature FIX prior to proteolytic activation), and was found to bind an epitope overlapping with the FIXa epitope bound by FVIIIa. The FX-binding arm, “BIIB-12-917”, reportedly showed selective binding to FX zymogen, lacked detectable binding to (activated) FXa, and bound an epitope of FX that lies within the activation peptide (which is present in FX zymogen but not FXa). Further mutations were then introduced into selected FIX-binding antibodies, including BUB-9-1336, to generate libraries from which to select for antibodies with even further increased specificity and/or affinity for FIXa.

WO2018/141863 and WO2018/145125 also described anti-FIXaxFX bispecific antibodies and their use as procoagulants for treating or reducing bleeding.

SUMMARY OF THE INVENTION

The present invention relates to improved bispecific antigen-binding molecules that bind blood clotting factors FIXa and FX. The bispecific antigen-binding molecules of the present invention enhance the FIXa-catalysed activation of FX to FXa, and can effectively replace the natural cofactor FVIIIa which is missing in patients with haemophilia A, to restore the ability of the patients' blood to clot. See FIG. 2.

As reported here, the inventors succeeded in generating a number of bispecific antigen-binding molecules having suitable qualities for development as therapeutic products, including very high potency in enhancing FX activation. Described are bispecific antigen-binding molecules having novel binding sites for anti-FIXa and anti-FX, which can be used to effectively substitute for FVIIIa in the blood clotting cascade. In particular, an anti-FIXa binding site is described which is highly active in combination with an array of different anti-FX binding sites and can thus be incorporated into a variety of different FIXa-FX bispecifics, providing flexibility for selection of bispecific antibodies with further desired characteristics such as ease of manufacture.

The inventors have designed bispecific antibodies which combine a potent FVIII mimetic activity (as indicated by high performance in in vitro assays) with robust biochemical and biophysical properties suitable for clinical scale manufacturing (including expression, bispecific molecular assembly, purification and formulation), and which are of fully human origin, thereby minimising the risk of immunogenicity in human in vivo therapy.

Aspects of the invention are set out in the appended claims, and further embodiments and preferred features of the invention are described below.

In a first aspect, the present invention relates to bispecific antigen-binding molecules comprising (i) a FIXa binding polypeptide arm comprising a FIXa binding site, and (ii) a FX binding polypeptide arm comprising a FX binding site. The FIXa and/or the FX binding polypeptide arm may comprise an antibody Fv region comprising the FIXa or FX binding site respectively. An antibody Fv region is an antibody VH-VL domain pair. The VH domain comprises HCDR1, HCDR2 and HCDR3 in a VH domain framework, and the VL domain comprises LCDR1, LCDR2 and LCDR3 in a VL domain framework. The polypeptide arm may comprise an antibody heavy chain (optionally one comprising an IgG constant region) and/or an antibody light chain.

Antigen-binding molecules of the present invention may thus comprise

first and second antibody Fv regions, the first and second antibody Fv regions comprising binding sites for FIXa and for FX respectively, and

a half-life extending region for prolonging the half-life of the molecule in vivo.

The half-life extending region may be a heterodimerisation region, comprising a first polypeptide covalently linked (e.g., as a fusion protein) to the first antibody Fv region and a second polypeptide covalently linked (e.g., as a fusion protein) to the second antibody Fv region, wherein the two polypeptides pair covalently and/or non-covalently with one another. The first and second polypeptides of the heterodimerisation region may have identical or different amino acid sequences. The heterodimerisation region may comprise one or more antibody constant domains, e.g., it may be an antibody Fc region.

Bispecific antigen-binding molecules of the present invention are able to bind FIXa through the FIXa binding site of the FIXa binding polypeptide arm and to bind FX through the FX binding site of the FX binding polypeptide arm, and thereby enhance the FIXa-catalysed activation of FX to FXa. This may be determined in an in vitro FX activation assay as described herein.

The FIXa binding site may be provided by a set of complementarity determining regions (CDRs) in the FIXa binding polypeptide arm, the set of CDRs comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3. Optionally, HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408. Optionally, LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7 and LCDR3 is SEQ ID NO: 8.

The set of HCDRs in the FIXa binding polypeptide arm may be the set of HCDRs of any anti-FIX VH domain shown herein, such as any shown in Table S-9A, any identified in Table N, or any of the VH domains N0128H, N0436H, N0511H, N1091H, N1172H, N1280H, N1314H, N1327H or N1333H shown in FIG. 20. HCDR1 may be SEQ ID NO: 441. HCDR2 may be SEQ ID NO: 634 or SEQ ID NO: 436. HCDR3 may be SEQ ID NO: 635 or SEQ ID NO: 433. The CDRs may be the N1280 CDRs, wherein HCDR1 is SEQ ID NO: 441, HCDR2 is SEQ ID NO: 436 and HCDR3 is SEQ ID NO: 533. Alternatively the CDRs may be the N1333H CDRs.

The set of LCDRs in the FIXa binding polypeptide arm may be the set of LCDRs of any anti-FIX VL domain shown herein. The LCDRs may be the LCDRs of 0128L as shown in Table S-50. LCDR1 may be SEQ ID NO: 6, LCDR2 may be SEQ ID NO: 7 and/or LCDR3 may be SEQ ID NO: 8.

Optionally, one or more amino acids in the set of CDRs may be mutated to differ from these sequences. For example, the set of CDRs may comprise 1, 2, 3, 4 or 5 amino acid alterations, the altered residue or residues being in any one or more of the heavy or light chain CDRs. For example the set of CDRs may comprise one or two conservative substitutions. The choice of mutations, e.g., substitutions, can be informed by the information and analysis provided in the Examples herein.

The FIXa binding polypeptide arm may comprise an antibody VH domain comprising a set of HCDRs HCDR1, HCDR2 and HCDR3. The sequence of HCDR1 may be SEQ ID NO: 406, optionally with one or two amino acid alterations (e.g., substitutions). The sequence of HCDR2 may be SEQ ID NO: 407, optionally with one or two amino acid alterations (e.g., substitutions). The sequence of HCDR3 may be SEQ ID NO: 408, optionally with one or two amino acid alterations (e.g., substitutions).

The FIXa binding polypeptide arm may comprise an antibody VL domain comprising a set of LCDRs LCDR1, LCDR2 and LCDR3. The sequence of LCDR1 may be SEQ ID NO: 6, optionally with one or two amino acid alterations (e.g., substitutions). The sequence of LCDR2 may be SEQ ID NO: 7, optionally with one or two amino acid alterations (e.g., substitutions). The sequence of LCDR3 may be SEQ ID NO: 8, optionally with one or two amino acid alterations (e.g., substitutions).

The antibody Fv region of the FIXa binding polypeptide arm may comprise a VH domain generated through recombination of immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is VH3-7 (e.g., VH3-7*01), wherein the j gene segment is JH6 (e.g. JH6*02), and optionally wherein the d gene segment is DH1-26 (e.g., DH1-26*01), and/or it may comprise a VL domain generated through recombination of immunoglobulin light chain v and j gene segments, wherein the v gene segment is VL3-21 (e.g., VL3-21*d01) and the j gene segment is JL2 (e.g., JL2*01). In another embodiment, a VL domain may be one that is generated through recombination of immunoglobulin light chain v and j gene segments, wherein the v gene segment is VL3-21 (e.g., VL3-21*d01) and the j gene segment is JL3 (e.g., JL3*02).

The amino acid sequence of the VH domain of a FIXa polypeptide binding arm may share at least 90% sequence identity with a VH domain shown in FIG. 20, e.g., the N1280H VH domain. Sequence identity may be at least 95%, at least 97%, at least 98% or at least 99%. Optionally the VH domain is one of the anti-FIX VH domains shown herein, such as any shown in Table S-9A, any identified in Table N, or any of the VH domains N0128H, N0436H, N0511H, N1091H, N1172H, N1280H, N1314H, N1327H or N1333H shown in FIG. 20. Optionally the VH domain is N1280H, N1333H, N1441, N1442 or N1454. Optionally the anti-FIXa VH domain comprises the amino acid sequence of any of said VH domains (e.g., N1280H) with up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions. Substitutions may optionally be in one or more framework regions, e.g., there may be 1 or 2 substitutions in FR3, optionally at IMGT position 84 and/or IMGT position 86.

The amino acid sequence of the VL domain may share at least 90% sequence identity with SEQ ID NO: 10 (0128L). Sequence identity may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Optionally the VL domain amino acid sequence is SEQ ID NO: 10. The VL domain amino acid sequence may alternatively be SEQ ID NO: 416.

The FX binding site may be provided by a set of CDRs in the FX binding polypeptide arm. The FX binding polypeptide arm may comprise an antibody VH-VL domain pair (i.e., an antibody Fv region), the VH domain comprising HCDR1, HCDR2 and HCDR3 in a framework, and the VL domain comprising LCDR1, LCDR2 and LCDR3 in a framework.

The FX binding site may be provided by the HCDRs of any anti-FX VH domain identified herein (e.g., any set of HCDR1, HCDR2 and HCDR3 of a VH domain shown in Table S10-C and/or in FIG. 11) and the 0128L LCDRs.

The FX binding polypeptide arm may comprise a VH domain having at least 90% amino acid sequence identity with a VH domain disclosed herein, including any in Table S-10C and/or in FIG. 11—for example the T0687H, T0736H or T0999H VH domain. Sequence identity may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Optionally the VH domain comprises the amino acid sequence of said VH domain with up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions. Substitutions may optionally be in one or more framework regions.

The FX binding polypeptide arm may comprise a VH domain having at least 90% amino acid sequence identity with the T0201 VH domain (shown in FIG. 11). Sequence identity may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Optionally the VH domain comprises the amino acid sequence of said VH domain with up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions. Substitutions may optionally be in one or more framework regions.

The FX binding polypeptide arm may comprise any VH domain amino acid sequence identified herein, such as any shown in Table S-10C, any identified in Table T or any from FIG. 11. Optionally the VH domain is T0201H, T0687H, T0736H or T0999H.

The FX binding polypeptide arm may comprise a VL domain having at least 90% amino acid sequence identity with the 0128L VL domain SEQ ID NO: 10. Sequence identity may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Optionally the VL domain comprises the amino acid sequence of the 0128L VL domain with up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions. Optionally the VL domain amino acid sequence is SEQ ID NO: 10. Alternatively the VL domain sequence is SEQ ID NO: 416.

The FX binding polypeptide arm may comprise an antibody Fv region comprising

a VH domain generated through recombination of immunoglobulin heavy chain v, d and j gene segments, wherein the v and j gene segments are IGHV1-46 (e.g., VH1-46*03) and IGHJ1 (e.g., JH1*01), and optionally wherein the d gene segment is IGHD6-6 (e.g., DH6-6*01), and

a VL domain generated through recombination of immunoglobulin light chain v and j gene segments, wherein the v and j gene segments are IGLV3-21 (e.g., VL3-21*d01) and IGLJ2 (e.g., JL2*01) or IGLJ3 (e.g., JL3*02).

Accordingly, one aspect of the present invention is a bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein

the first VH domain comprises a set of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408, and/or wherein the first VH domain is at least 95% identical to the N1280H VH domain at the amino acid sequence level;

the second VH domain is at least 95% identical to the T0201H VH domain at the amino acid sequence level, and

the first VL domain and the second VL domain each comprise a set of LCDRs comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences defined wherein LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7 and LCDR3 is SEQ ID NO: 8, and/or wherein the first VL domain and the second VL domain are at least 95% identical to the 0128L VL domain SEQ ID NO: 10 at the amino acid sequence level.

Another aspect of the present invention is a bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein

the first VH domain is a product of recombination of human immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is IGHV3-7 (e.g., VH3-7*01) and the j gene segment is IGHJ6 (e.g., JH6*02),

the second VH domain is a product of recombination of human immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is IGHV1-46 (e.g., VH1-46*03) and the j gene segment is IGHJ1 (e.g., JH1*01), and optionally wherein the d gene segment is IGHD6-6 (e.g., DH6-6*01), and

the first VL domain and the second VL domain are both products of recombination of human immunoglobulin light chain v and j gene segments, wherein the v gene segment is IGLV3-21 (e.g., VL3-21*d01) and the j gene segment is IGLJ2 (e.g., JL2*01) or IGLJ3 (e.g., JL3*02).

Another aspect of the present invention is a bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein

the first VH domain has at least 95% amino acid sequence identity with the N1280H VH domain,

the second VH domain has at least 95% amino acid sequence identity with the T0201H VH domain, and

the first VL domain and the second VL domain each have at least 95% amino acid sequence identity with the 0128L VL domain.

The first VH domain may comprise a set of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408.

The first VH domain may have at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1280H. The first VH domain may comprise a set of N1280H HCDRs comprising N1280H HCDR1, N1280H HCDR2 and N1280H HCDR3. For example, it may be the N1280H VH domain. Alternatively, the VH domain may be the N1441H, N1442H or N1454H VH domain.

Amino acid sequences of example VH domains and sets of VH CDRs are shown in FIG. 20 and/or in Table S-9A. The first VH domain of the bispecific antibody may be, or may have at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to, any of these VH domains. Optionally it may comprise a VH domain amino acid sequence having up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions compared with said VH domain. Substitutions are optionally in framework regions.

Examples of residues and substitutions that may be retained or introduced in the first VH domain include the following (defined with reference to N1280H, with IMGT numbering as shown in FIG. 14):

Substitution of another residue (e.g., Asp, Glu, His, Asn, Gln, Met, Thr, Gly, Ser, Ala, Ile, Leu, Val or Tyr) at Lys84 in FR3, e.g., Lys84Asp or Lys84Glu; and

Substitution of another residue at Ser86 in FR3, e.g., a negatively charged residue such as Glu (Ser86Glu).

Further examples include:

Substitution of a negatively charged residue (e.g., Asp or Glu) or His at one or more of Gln3, Val5, Gly9, Gly11, Gly16, Gly17 and Leu21 FR1, such as any of Gln3Asp, Gln3Glu, Gln3His, Val5Glu, Gly9Glu, Gly11Asp, Gly11Glu, Gly11His, Gly16Glu, Gly17Asp, Gly17Glu or Leu21Asp;

Substitution of a negatively charged residue at Val68 and/or Val71 in FR3, e.g., Val68Asp, Val68Glu or Val71Glu;

Substitution of His, Gln or Leu at Arg75 in FR3;

Substitution of Ser, Thr, Gly, Leu or Lys at Arg80 in FR3; and

Substitution of Asp or His at Asn82 in FR3.

Any one or more of the above-listed sequence features may be included.

The second VH domain may be, or may have at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to T0201H or any other VH domain shown in FIG. 11 and/or in Table S-10C. Optionally it may comprise a VH domain amino acid sequence having up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions compared with said VH domain. Substitutions are optionally in framework regions. The second VH domain may comprise an HCDR1 which is the T0201H HCDR1, an HCDR2 which is the T0201H HCDR1, and/or an HCDR3 which is the T0201H HCDR3. Amino acid sequences of these CDRs are shown in FIGS. 11 and 12 and in Table S-10C. Further example CDRs are indicated in FIG. 12 and in Table T. For example, the second VH domain may comprise:

an HCDR1 which is the T0201 HCDR1 or the T0736 HCDR1,

an HCDR2 which is the T0201 HCDR2, and/or

an HCDR3 which is the T0201 HCDR3, the T0687 HCDR3 or the T0736 HCDR3.

Optionally, HCDR1 is SEQ ID NO: 636 or SEQ ID NO: 598. Optionally, HCDR2 is SEQ ID NO: 467. Optionally, HCDR3 is SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639 or SEQ ID NO: 565.

Examples of residues and substitutions that may be retained or introduced in the second VH domain include the following (defined with reference to T0201H, with IMGT numbering as shown in FIG. 13):

Substitution of another amino acid residue at Cys114, e.g., wherein the substituted residue is Ile, Gln, Arg, Val or Trp, (preferably Ile, Val or Leu);

Substitution of another positively charged residue for Gln3 in FR1, e.g., Gln3Arg or Gln3Lys;

Germlining of residues in framework regions, e.g., Ile5Val (substitution of valine for isoleucine at residue 5 in FR1), or replacement of a non-germline residue in a framework region by a different non-germline residue, e.g., Ile5Arg (substitution of arginine for isoleucine at residue 5 in FR1);

Substitution of another amino acid residue (e.g., Lys, Ala or Gly) at Glu11 in FR1, e.g., Glu11Lys;

Substitution of a positively charged amino acid residue for Gly16 in FR1, e.g., Gly16Arg;

Presence of Met at position 39 in FR2, or alternatively Leu at this position;

Presence of Ser at position 62 and/or position 64 in CDR2;

Substitution of Tyr at Phe71 in FR3;

Substitution of a positively charged residue at Thr82 in FR3, e.g., Thr82Arg or Thr82Lys; Presence of Ser at position 85 in FR3, or alternatively Thr at this position;

Substitution of a positively charged residue at Thr86 in FR3, e.g., Thr86Arg or Thr86Lys.

Any one or more of the above-listed sequence features may be included.

The FIXa binding polypeptide arm and the FX binding polypeptide arm may each comprise an antibody Fv, wherein the VL domain of each Fv has an identical amino acid sequence, i.e. the bispecific antigen-binding molecule has a common VL domain. The molecule may have a common light chain comprising a variable region and a constant region, optionally a human lambda constant region.

The bispecific antigen-binding molecule may be a tetrameric immunoglobulin comprising

a first pair of antibody heavy and light chains (heavy-light chain pair) comprising a FIXa binding Fv region,

a second heavy-light chain pair comprising a FX binding Fv region,

wherein each heavy chain comprises a VH domain and a constant region, and each light chain comprises a VL domain and a constant region, and wherein the first and second heavy-light chain pairs associate through heterodimerisation of their heavy chain constant regions to form the immunoglobulin tetramer.

As noted, the light chain may be a common light chain, i.e., the light chain of the first and second heavy-light chain pairs has an identical amino acid sequence. Each heavy-light chain pair may comprise the 0128L CL constant domain paired with a CH1 domain. The sequence of the light chain may be SEQ ID NO: 405. Alternatively the sequence of the light chain may be SEQ ID NO: 414. Exemplary immunoglobulin isotypes include human IgG, e.g., IgG4, optionally with engineered constant domains such as IgG4 PE.

The Fc domain of a bispecific antibody may be engineered to promote heterodimerisation over homodimerization. For example, the heavy chain constant region of the first heavy-light chain pair may comprise a different amino acid sequence from the heavy chain constant region of the second heavy-light chain pair, wherein the different amino acid sequences are engineered to promote heterodimerisation of the heavy chain constant regions. Examples include knobs-into-holes mutations or charge pair mutations. Alternatively, the heavy chain constant region of the first heavy-light chain pair may be identical to the heavy chain constant region of the second heavy-light chain pair, in which case it is expected that both homodimers and heterodimers will assemble, and these will be subsequently separated using one or more purification steps in the antibody manufacturing process to isolate the desired heterodimer comprising one anti-FIXa arm and one anti-FX arm.

An advantageous feature of bispecific antibodies exemplified here is that they have been generated from human immunoglobulin gene segments, using the Kymouse platform. Unlike antibodies generated from immunisation of normal laboratory animals, which may require “humanisation” steps such as grafting of mouse CDRs into human antibody variable domains and iterative refinement of the engineered variable domains to mitigate a loss of function resulting from these changes, the antibodies of the present invention were generated and selected from the outset with fully human antibody variable domains. The use of a fully human antibody is of special relevance in the context of haemophilia treatment, where low immunogenicity is paramount, as noted above. The low immunogenicity of the bispecific antibodies of the present invention renders them suitable for treatment of haemophilia A patients, including those with or without inhibitory antibodies to other treatments such as FVIII. Patients receiving antigen-binding molecules of the present invention should be at minimal risk of developing an immunogenic response to the therapy.

The mode of action of the bispecific molecules is also associated with a good safety profile, with low risk of complications such as deep vein thrombosis and pulmonary embolism. Activity of the bispecific molecules is comparable with that of natural FVIII and a mechanism of action that is integrated within the existing blood coagulation pathway, being activated only in the context of upstream triggering of the natural clotting cascade.

Bispecific antibodies according to the present invention have shown strong activity in a number of functionally relevant assays for FVIII mimetic activity, including factor Xase assay, activated partial thromboplastin time (aPTT) assay and thrombin generation assay (TGA), as exemplified herein.

Other desirable features include long-half life (reducing the required frequency of administration) and amenability of the molecules to formulation at high concentration (facilitating subcutaneous injection in the home setting).

Patient compliance is recognised to be a significant issue for long term self-administered therapy, especially among teenage and young adult patients. For a treatment to succeed in the field, its administration schedule should be simple for the patient to understand and follow with minimum inconvenience. Long intervals between administered doses are desirable, but reducing dose frequency without sacrificing therapeutic activity requires a product with both a long in vivo half life and a sufficient efficacy at “trough” concentrations towards the end of a dosing period. Antigen-binding molecules according to the present invention desirably have a long in vivo half life. This can be facilitated by inclusion of an Fc region which undergoes recycling in vivo via FcRn. Antigen-binding molecules according to the present invention also preferably maintain high functional activity at low concentration. We found that bispecific antibodies according to the present invention have a thrombogenic activity similar to that of emicizumab but with an increase in thrombogenic activity that is most pronounced at lower concentrations. Data disclosed herein indicate that bispecific antibodies according to the present invention possess a thrombogenic activity that is the same as or surpasses that of emicizumab at concentrations in at least the range of 1 to 300 nM, for example when the antibody and emicizumab are tested at the following concentrations:

1-30 nM, e.g., at 1 nM, at 3 nM, at 10 nM and/or at 30 nM;

100-300 nM, e.g., at 100 nM and/or at 300 nM.

Activity can be measured in the thrombin generation assay described herein. Effective activity at low concentrations may help to ensure that protection against bleeds is maintained towards the end of a dosing period—the in vivo concentration of the antibody being lowest in the final days before the next dose is due. It may also assist in protecting areas of the body which are relatively poorly perfused by the circulation—including the joints, which are a common site of problematic bleeding in haemophiliac patients.

Further aspects of the invention relate to pharmaceutical compositions comprising the bispecific antigen-binding molecules and their use in medicine including for the treatment of haemophilia A, as set out in the appended claims and described in the present disclosure.

Monospecific antibodies are also provided as aspects of the present invention. Thus, an anti-FIXa antibody may comprise two copies of a first heavy-light chain pair as defined herein. An anti-FX antibody may comprise two copies of a second heavy-light chain pair as defined herein.

Further aspects include nucleic acid molecules encoding sequences of the antibodies described herein, host cells containing such nucleic acids, and methods of producing the antibodies by culturing the host cells and expressing and optionally isolating or purifying the antibodies. The expressed antibody is thereby obtained. VH and VL domains of antibodies described herein may similarly be produced and are aspects of the present invention. Suitable production methods of antibodies include large-scale expression from host cells (e.g, mammalian cells) in a bioreactor by continuous or batch culture (e.g., fed batch culture).

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects and embodiments of the invention will now be described in more detail, with reference to the drawings, in which:

FIG. 1 illustrates the blood coagulation cascade [6].

FIG. 2 shows (A) co-factor action of FVIIIa interacting with FIXa and FX; (B) co-factor action of bispecific antibody interacting with FIXa and FX; and (C) bispecific antibody interacting with FIXa (9a) and FX (10) on the surface of a platelet. Bispecific antibody in this embodiment is a four chain molecule having two disulphide-linked heavy chains each comprising (N to C) domains VH1-CH1-CH2-CH3 and two identical light chains (common light chain) comprising (N to C) domains VL-CL. In this illustration, binding site comprising VH1-VL binds to FX and binding site comprising VH2-VL binds to FXa.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 334) of factor IX, with residue numbering for the mature protein. The signal peptide (straight underlined) is cleaved after secretion. The propeptide (wave underlined) is cleaved on maturation. Mature factor IX contains a light chain (residues 1-145) and a heavy chain (residues 146-415). The activation peptide (boxed) is cleaved on activation, generating activated factor IXa which contains a light chain (residues 1-145) and a heavy chain (residues 181-415, bold) joined by a disulphide bridge between Cys132 and Cys289.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 335) of factor X, with residue numbering. Residues 1-31 are a signal peptide (straight underlined). Residues 32-40 are a propeptide (wave underlined). The FX light chain is residues 41-179. The FX heavy chain is residues 183-488. The FXa heavy chain is residues 235-488 (bold).

FIG. 5 shows an embodiment of the invention: bispecific IgG with common light chain.

FIG. 6 summarises the process of optimising the anti-FIXa VH domain sequence N0436H and the anti-FX VH domain sequence T0200 for functional combination with each other and for pairing with the N0128L common VL domain.

FIG. 7 illustrates (A) principles of in vitro assay for FVIII mimetic activity of a bispecific molecule (FXase or tenase assay); and (B) example data from the assay showing positive result for FIXa-FX bispecific molecule compared with negative control.

FIG. 8 shows the results of screening bispecific antibodies having various anti-FX arms in the FXase assay (standard reaction conditions). The bispecific antibody panel comprises a range of anti-FX VH test domains, each in combination with the N0128H anti-FIX VH domain and 0128L common VL domain.

FIG. 9 illustrates the B cell cluster identified for the lineage of the anti-FX T0200H domain.

FIG. 10 shows the results of screening optimised bispecific antibodies in the FXase assay. (A) FXase activity for IgG4 bispecific antibodies comprising named anti-FX VH domain combined with the N0128H, N1172H or N1280H anti-FIX VH domain and 0128L common light chain. OD at 405 nm at 10 minutes (600 s). (B) FXase activity for IgG4 bispecific antibodies comprising named anti-FX VH domain combined with the N1280H anti-FIX VH domain and N0128L common light chain.

FIG. 11 is an amino acid sequence alignment of a selection of anti-FX VH domains from the T0200H B cell cluster. Germline sequence (SEQ ID NO: 518) is shown for comparison. CDRs are boxed.

FIG. 12 identifies mutants of the T0201H VH domain in which the terminal four residues of CDR3 were individually mutated to other amino acids. For example the T0590H VH domain is a Cys114Ala mutant of the T0201H VH domain, i.e., in which the cysteine at IMGT position 114 is replaced by alanine.

FIG. 13 shows the amino acid sequence of VH domain T0201H, annotated in (A) to show FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and in (B) to show IMGT numbering.

FIG. 14 shows the amino acid sequence of VH domain N0128H, aligned against N192H and N1280H and annotated in (A) to show FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and in (B) to show IMGT numbering.

FIG. 15 shows the kinetics of activity of (A) IXAX-1280.0201.0128 and of (B) the comparator antibody AbE in the FXase assay. Antibody was purified by Protein A and is composed of a homodimer/heterodimer mixture.

FIG. 16 shows results of bispecific antibodies in Xase assay. “E” is Antibody E positive control. FXase activities are plotted as OD405 nm at 10 minutes for samples normalised to 12.5 μg/mL (10.4 nM) final concentration. Bispecific antibodies are ranked in order of activity.

FIG. 17 shows the effect of bispecific antibody optimisation on FVIII mimetic activity (FXase). Results are shown for of bispecific antibodies, each having the named VH domain in the anti-FIXa arm and T0638H VH domain in the anti-FX arm, both paired with the 0128L common light chain VL domain. FVIII mimetic activity (FXase) was measured using an in vitro chromogenic Factor X activation assay at 405 nm (Y-axis) as a function of time in seconds (X-axis). Optimised bispecific antibodies are individually labelled. A human IgG4 isotype control demonstrates no FVIII mimetic activity. The key shows the name of the VH domain in the antibody to the right of its corresponding graph in order of maximal FXase activity, i.e., N1333H>N1280H>N1172H>N1091H>N0511H>N0436H>N0128H>control.

FIG. 18 shows aPTT assay results for IgG4 bispecific antibodies comprising named anti-FX VH domain combined with the N0128H, N1172H or N1280H anti-FIX VH domain and 0128L common light chain.

FIG. 19 shows a dose response for IXAX-1280.0201.0128 bispecific antibody (purified on Protein A) in a one-stage activated aPTT clotting assay using FVIII-depleted human plasma.

FIG. 20 shows VH domain amino acid sequences of N0128H, N0436H, N0511H, N1091H, N1172H, N1280H, N1314H, N1327H and N1333H, annotated to identify their framework regions and CDRs.

FIG. 21 is an SPR sensorgram of bispecific antibody binding to antigen, where simultaneous binding to FIX and FX is demonstrated, compared with sensorgram of (A) isotype control antibody or (B) monospecific antibody.

FIG. 22 presents summary results of FXase assays for selected CDR1 and CDR2 sequence variants of T0681H. Except where indicated for AbE controls, samples were purified by Protein A chromatography and their final concentrations normalised to 12.5 μg/mL (10.4 nM). 3 samples of AbE positive control antibody are included: (i) protein A purified; 12.5 μg/mL; (ii) protein A purified; 37.5 μg/mL; (iii) protein A purified followed by further purification by ion exchange chromatography; 37.5 μg/mL. All data shown were sampled at the 10 minute time-point.

FIG. 23 summarises the effect of T0201H CDR1, CDR2 and CDR3 mutagenesis on bispecific antibody FXase activity in vitro. Bispecific antibody anti-FX T0201H variant VH domains were expressed with anti-FIX N01280H arm and N128_IgL common light chain in HEK cells, purified by Protein A chromatography and normalised by concentration to 0.15 mg/ml (125 nM final concentration). In vitro FXase assay was performed using 5 μl of purified material. Data shown are at 10 minute time point. Dotted line represents FXase activity of T0201H.

FIG. 24 presents summary results of aPTT clotting assays investigating the effect of sequence variation in the anti-FX T0201H VH domain on bispecific antibody activity. Clotting time (seconds) was recorded after spiking FVIII deficient plasma with bispecific antibodies comprising the named anti-FX VH arm, anti-FIX N01280H arm and N128_IgL common light chain. Bispecific antibodies were expressed in HEK cells, purified by Protein A chromatography and analysed at three different concentrations 0.1 mg/ml, 0.3 mg/ml and 0.5 mg/ml. Dotted lines indicate the clotting times of FVIII deficient plasma spiked with a human IgG4 isotype control or normal pooled human plasma spiked with PBS.

FIG. 25 identifies CDRs of VH domains which were progressively improved for FVIII mimetic activity during the mutagenesis process. Black shading identifies VH domains better than those in the same sub-table.

FIG. 26 shows the thrombin generation curve of normal pooled plasma using factor IXa as a trigger in a thrombin generation assay (TGA). The thrombogram depicts the generation of thrombin over time in sample plasma, plotted as the concentration (nM) of thrombin generated during clotting over time (minutes).

FIG. 27 describes the thrombin generation of FVIII deficient plasma spiked with bispecific antibodies at final concentration of 133 nM comprising CDR1, CDR2 and CDR3 single and combinatorial variants of T0201H, compared with AbE, isotype control, normal plasma and emicizumab calibrator. FIXa trigger at 0.3 nM. (A) 80 minute thrombogram. (B) 25 minute thrombogram.

FIG. 28 shows thrombin Cmax (nM) and Tmax (min) from TGA carried out on FVIII deficient plasma spiked with bispecific antibodies comprising CDR1, CDR2 and CDR3 single and combinatorial mutants of T0201H, compared with AbE, isotype control, normal plasma and emicizumab calibrator. Test antibodies were purified by Protein A chromatography only and therefore represent a mixture of heterodimer and homodimer. (A) Antibody concentration 133 nM. (B) Antibody concentration 80 nM.

FIG. 29 illustrates Cmax (nM) and Tmax (min) dose responses from TGA carried out on FVIII deficient plasma spiked with (A) bispecific antibody IXAX-1280.0999.0128 and (B) emicizumab calibrator. Isotype control (plus sign) used was a human IgG4 and was spiked into FVIII deficient plasma. Normal pooled plasma (cross sign) was spiked with PBS.

FIG. 30 shows dose response curves for Cmax of IXAX-1280.0999.0325 and AbE in TGA. Vertical dotted lines indicate final antibody concentration corresponding to 4.5 μg/ml (30 nM), 10 μg/ml (66.6 nM) and 45 μg/ml (300 nM). Horizontal lines represent Cmax of normal pooled plasma collected from healthy volunteers.

FIG. 31 shows thrombograms of (A) IXAX-1280.0999.0325 “BiAb_1” and (B) AbE, in human FVIII-depleted plasma, at antibody concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 300 nM. Thrombogram of a normal human plasma sample is shown in shadow.

FIG. 32 shows dose response curves for Tmax of IXAX-1280.0999.0325 and AbE in TGA. Vertical dotted lines indicate antibody concentration corresponding to 4.5 μg/ml (30 nM), 10 μg/ml (66.6 nM) and 45 μg/ml (300 nM). Horizontal lines represent Tmax of normal pooled plasma collected from healthy volunteers.

FIG. 33 shows thrombin generation abilities in terms of (A) Cmax and (B) Tmax of BiAb_1 (IXAX-1280.0999.0325), BiAb_2 (IXAX-1454.0999.0325), BiAb_3 (IXAX-1441.0999.0325) and BiAb 4 (IXAX-1442.0736.0325) compared with commercially available emicizumab calibrator in a TGA assay in commercially available human FVIII-depleted plasma.

FIG. 34 shows thrombin generation abilities in terms of (A) Cmax and (B) Tmax of BiAb_1 (IXAX-1280.0999.0325) compared with commercially available emicizumab calibrator in a TGA assay in human FVIII-depleted plasma.

FIG. 35 shows purification yield and % heterodimer for bispecific antibody IXAX-1172.0201.0128 expressed from 8 independent minipools of stably transfected CHO cells.

FIG. 36 plots the correlation between bispecific antibody activity by FXase assay (activity at 10 min) and % heterodimer for bispecific antibody IXAX-1172.0201.0128 normalised to 0.3 mg/ml expressed from 8 independent minipools. Pearson's correlation coefficient was calculated as 0.9939.

FIG. 37 (A) shows separation of IXAX-1280.0999.0128 by ion exchange chromatography on a 1 ml CaptoSP ImpRes column and a linear NaCl gradient up to 500 nM in 20 nM sodium phosphate, pH 6.0. Absorbance, mAU (milli absorbance unit); conductivity, mS/cm (milli Siemens per centimetre). Peak 1 is NINA-1280.0128 monospecific anti-FIX antibody. Peak 2 is IXAX-1280.0999.0128 bispecific antibody. Peak 3 is TINA-0999.0128 monospecific anti-FX antibody. (B) shows separation of IXAX-0436.0202.0128 by ion exchange chromatography with stepwise elution. Peak 1 is NINA-0436.0128 monospecific anti-FIX antibody. Peak 2 is IXAX-0436.0202.0128 bispecific antibody. Peak 3 is TINA-0202.0128 moospecific anti-FX antibody. (C) shows separation of IXAX-1172.0201.0128 by ion exchange chromatography. Peak 1 is NINA-1172.0128 anti-FIX monospecific antibody. Peak 2 is IXAX-1172.0201.0128 bispecific antibody.

FIG. 38 shows cation exchange purification of purified FIX/FX heterodimers for each of bispecific antibodies (A) IXAX-1280.0999.0325 (B) IXAX-1454.0999.0325 (C) IXAX-1441.0999.0325 and (D) IXAX-1442.0736.0325.

FIG. 39 shows dose response in the FXase assay with IXAX-1280.0999.0325 and AbE. Dotted lines indicate antibody concentration corresponding to 4.5 μg/ml, 10 μg/ml and 45 μg/ml.

FIG. 40 shows dose response in a chromogenic FVIII mimetic activity Hyphen assay with IXAX-1280.0999.0325 and AbE.

FIG. 41 shows dose response in the aPTT assay with IXAX-1280.0999.0325 and AbE. Vertical dotted lines represent 66.6 nM (10 μg/ml) and 300 nM (45 μg/ml) final antibody concentration; horizontal lines represent aPTT value of normal pooled plasma collected from healthy volunteers.

FIG. 42 presents results of aPTT clotting time assays investigating the effect of bispecific antibodies IXAX-1280.0999.0325 (circle), IXAX-1441.0999.0325 (diamond) and AbE (cross) in inhibitor plasma. Dose responses are shown for these antibodies in a one-stage aPTT clotting assay using plasma obtained from a patient with haemophilia A demonstrating a specific inhibitor level of 70 BU to FVIII. Dotted horizontal line indicates the clotting time of the inhibitor plasma spiked with a human IgG4 isotype control.

FIG. 43 shows a thrombin peak height (Cmax) dose response for IgG4 bispecific antibodies IXAX-1280.0999.0325, IXAX-1441.0999.0325 and AbE (all purified on Protein A, followed by cation exchange chromatography) in a thrombin generation assay with plasma obtained from a patient with haemophilia A demonstrating a specific inhibitor level of 70 BU to FVIII. Bispecific antibody concentrations in nM are indicated for each dilution.

FIG. 44 shows a dose response for IgG4 bispecific antibodies (A) IXAX-1280.0999.0325, (B) IXAX-1441.0999.0325 and (C) AbE (all purified on Protein A, followed by cation exchange chromatography) in a thrombin generation assay with plasma obtained from a patient with haemophilia A demonstrating a specific inhibitor level of 70 BU to FVIII. Bispecific antibody concentrations analysed are 100, 33.3, 11.1, 3.7 and 1.23 nM. Grey shaded area indicates thrombin generation of normal pooled plasma. TGA trigger is FIXa.

DETAILED DESCRIPTION Blood Coagulation

The blood coagulation cascade is diagrammed in FIG. 1. Coagulation or clotting is one of the most important biological processes which stops blood loss from a damaged vessel to allow the vessel to be repaired. The mechanism of coagulation involves activation, adhesion, and aggregation of platelets along with deposition and maturation of fibrin. Misregulation of coagulation can result in excessive bleeding (haemophilia) or obstructive clotting (thrombosis). Coagulation is highly conserved in all mammals. It is controlled by a complex network of coagulation factors. Coagulation is initiated when the endothelium lining the blood vessel is damaged. The exposure of subendothelial tissue factor (TF) to plasma factor VII (FVII) leads to primary haemostasis (extrinsic pathway): a loose plug is formed at the site of injury. Activation of additional coagulation factors, especially factor IX (FIX) and factor VIII (FVIII), leads to secondary haemostasis (intrinsic pathway): fibrin strands are formed to strengthen the plug. Extrinsic and intrinsic pathways ultimately converge to a common point: the formation of the factor Xa/Va complex which together with calcium and bound on a phospholipid surface generate thrombin (factor IIa) from prothrombin (factor II).

FVIII is cleaved by thrombin or factor Xa (FXa), and the resultant factor Villa (FVIIIa) presents a heterotrimeric structure consisting of the A1 subunit, the A2 subunit, and the light chain. Upon activation and in the presence of calcium ions and a phospholipid surface (on platelets), FVIIIa binds via its light chain and A2 subunit to FIXa and simultaneously binds via its A1 subunit to FX, forming an active intrinsic “tenase” or “Xase” complex in which the FVIIIa cofactor brings FIXa and FX into proximity and also allosterically enhances the catalytic rate constant of FIXa. See FIG. 2a . Factor X is activated by the serine protease activity of FIXa, and the clotting cascade continues, culminating in the deposition of fibrin, the structural polymer of the blood clot.

Haemophilia arise through a deficiency in the Xase complex, due either to a lack of FVIII cofactor activity (haemophilia A) or a lack of FIX enzyme activity (haemophilia B).

Factor IX (FIX)

Factor IX is a serine protease which requires factor VIII as a cofactor. It circulates in blood as an inactive precursor, which is activated through intrinsic or extrinsic pathway at the time of haemostatic challenge, as discussed above.

Unless the context requires otherwise, factor IX referred to herein is human factor IX, and factor IXa is human factor IXa.

The amino acid sequence of human factor IX is shown in FIG. 3. The factor IX gene is approximately 34 kb in length and contains 8 exons. The transcript comprises a short 5′ untranslated region, an open reading frame plus stop codon and a 3′ untranslated region. The ORF encodes a 461 amino acid pre-pro-protein in which the pre-sequence (signal peptide) directs factor IX for secretion, the propeptide sequence provides a binding domain for a vitamin K dependent carboxylase, which carboxylates certain glutamic acid residues in the adjacent GLA domain, and the remainder represents the factor IX zymogen, which enters into circulation after removal of the pre- and pro-sequences. The mature 415 residue FIX protein contains, from N to C terminus: a GLA domain in which 12 glutamic acid residues are post-translationally γ-carboxylated, two epidermal growth factor (EGF)-like domains, an activation peptide sequence and a catalytic serine protease domain. FIX is activated by either activated factor XI generated through the intrinsic pathway, or by the TF/FVIIa complex of the extrinsic pathway. Either way, activation involves cleavage of the peptide bond following R145 (α-cleavage) and of the peptide bond following R180 (β-cleavage), releasing an activation peptide corresponding to the intervening sequence, and thereby generating the activated FIXa molecule, which has an N terminal light chain (GLA-EGF-EGF) and a C terminal heavy chain (catalytic domain) joined by a disulphide bridge between C132 of the light chain and C289 of the heavy chain. Residue numbering refers to amino acids in the mature FIX polypeptide sequence. On the phospholipid surface where the Xase complex forms, it is the GLA domain of FIXa which associates with the phospholipid, while the catalytic domain stands high (>70 Å) above the phospholipid surface and is modulated by the A2 domain of FVIIIa [7, 8].

The molecular basis of haemophilia B—deficiency in FIXa activity—is diverse, including a variety of point mutations, nonsense mutations, mRNA splice site mutations, deletions, insertions, or mis-sense mutations at activation cleavage sites [9].

The catalytic (protease) domain of activated FIX (FIXa) is involved in binding to FVIIIa. Residue E245 in this domain binds calcium ions, and mutations at this position may reduce binding to FVIII and lead to haemophilia B, for example the substitution E245V. Mutations within the FIX helix formed by residues 330-338 are also linked with reduced binding to FVIII and consequently to haemophilia B.

Non-pathogenic mutations in factor IX have also been reported, including single nucleotide polymorphisms (SNPs) and length polymorphisms—reviewed in [9]. These include the MnII SNP in exon 6, resulting in T/A substitution at residue 148 (Malmö polymorphism), which is relatively common among white and black American populations [9].

Factor X (FX)

Unless the context requires otherwise, factor X referred to herein is human factor X, and factor Xa is human factor Xa. The amino acid sequence of human FX is shown in FIG. 4.

FX is also known as Stuart-Prower factor. It is a serine endopeptidase. FX can be activated, by hydrolysis, into factor Xa by either factor IX (together with its cofactor, factor FVIII, as described above) or factor VII (with its cofactor, tissue factor). FX acts by cleaving prothrombin in two places—at an Arg-Thr bond and then at an Arg-Ile bond, to yield the active thrombin.

Antigen-Binding

A desirable feature of the bispecific antigen-binding molecule is that it binds FIXa and FX in a manner that allows the bound FIXa to activate the bound FX.

To bring FIXa and FX together and thereby promote the activation of FX by FIXa, the bispecific antigen-binding molecule may bind these two cofactors simultaneously. Binding may occur sequentially, e.g., an initial binary complex may form between a first binding arm and its cognate antigen, followed by binding of the second binding arm to its cognate antigen. In principle these two binding events may occur in either sequence, i.e., FIXa followed by FX, or FX followed by FIXa. The molecular choreography is influenced by the relative affinities of the two binding sites for their respective antigens. In a population of bispecific antigen-binding molecules, FIXa and FX, a number of different complexes are expected to exist in parallel. Thus the pool will comprise free antigen-binding molecule, free FIXa, free FX, FIXa complexed with antigen-binding molecule, FX complexed with antigen-binding molecule, and a tertiary complex of FIX, FX and antigen-binding molecule, with each of these species being present in different proportions according to the relative on-rates and off-rates of the individual interactions.

It may be preferable for a bispecific antigen-binding molecule to have a higher affinity for FIXa than for FX. Such a bispecific molecule would be envisaged to form an initial complex with FIXa, which in turn would bind and activate FX. The relatively low affinity for FX reduces the proportion of FX that is bound in incomplete antibody-antigen complexes (i.e., without FIXa). A potential advantage of this is that it allows a greater proportion of FX to remain free to engage with any FVIII that may be present in a patient's blood. Haemophilia A encompasses a range of deficiencies in FVIII, ranging from mild deficiency to total absence of functional FVIII. For those patients who retain some functional FVIII, it may be desirable to retain this natural activity as far as possible. Thus, it may be desirable to provide a bispecific antigen-binding molecule in which the FX binding arm does not compete with FVIII for binding to FX.

Preferably the FX binding arm has a higher affinity for FX than for FXa. A low affinity for FXa promotes release of the activated product, completing the role of the FVIII-mimetic molecule in the coagulation cascade and freeing the FX binding site for re-use. In various embodiments, a bispecific described herein (e.g., antibody IXAX-1280.0999.0325 or antibody IXAX-1441.0999.0325), the FX binding arm of such a bispecific (e.g., binding arm comprising T0999H VH domain), or an anti-FX monospecific antibody comprising a homodimer of two such arms, has at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 10-fold higher, at least 100-fold higher affinity for FX than for FXa, e.g., at least 1000-fold higher affinity for FX than for FXa, and optionally does not show significant binding to FXa, e.g., as measured by ELISA. For example, in various embodiments the bispecific, FX binding arm or anti-FX monospecific antibody (e.g., TINA-0999.0325) does not bind human FXa as determined by ELISA and with reference to a negative control IgG. As an alternative to ELISA, affinity may be measured by SPR and the affinity for FX compared with affinity for FXa.

FIXa Binding

The FIXa binding arm of a bispecific antigen-binding molecule may bind the light chain and/or the heavy chain of FIXa. Initial studies indicated that FIXa binding arms of the N128 lineage described in the Examples do not bind the FIXa light chain in isolation (in the absence of the heavy chain).

A bispecific antigen-binding molecule of the present invention (or FIXa binding polypeptide arm thereof) may thus be one which binds a FIXa molecule comprising a heavy chain and a light chain, and which does not bind the FIX light chain in the absence of the heavy chain. Optionally, the FIXa binding arm recognises an epitope formed by, or stabilised by, the combination of the FIXa heavy and light chains. It may for example make contact only with the light chain in the FIXa molecule, binding an epitope that is exposed or stabilised only when the light chain is present in combination with the heavy chain in the FIXa molecule. Alternatively, it may contact an epitope comprising one or more residues from both the light chain and the heavy chain, or comprising residues of the heavy chain alone.

An antigen-binding molecule according to the present invention, or a FIXa-binding polypeptide arm thereof, may bind the EC domain of human FIXa with an affinity (measured as K_(D)) of 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. For example, K_(D) may be between 1 nM and 3 μM.

The K_(D) for binding human FIXa may be between 0.1 μM and 1 μM, e.g., between 0.15 and 0.3 μM. The K_(D) may be 0.6 μM or less, 0.5 μM or less, 0.4 μM or less, 0.3 μM or less, 0.25 μM or less, or 2 μM or less. The K_(D) may be at least 0.1 μM, for example at least 0.2 μM. It may be 0.1 μM-0.5 μM.

The K_(D) may be between 10 and 100 nM, e.g., between 25 and 75 nM.

The K_(D) may be 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. The K_(D) may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. The K_(D) may be at least 0.001 nM, for example at least 0.01 nM or at least 0.1 nM. The K_(D) may be between 0.1-10 nM.

An antigen-binding molecule according to the present invention, or a FIXa-binding polypeptide arm thereof, may bind human FIX with an affinity (measured as K_(D)) between 0.1 μM and 1 μM, e.g., between 0.15 and 0.3 μM. The K_(D) may be 0.6 μM or less, 0.5 μM or less, 0.4 μM or less, 0.3 μM or less, 0.25 μM or less, or 2 μM or less. The K_(D) may be at least 0.1 μM, for example at least 0.2 μM.

The K_(D) of interaction with FIX may be comparable to the K_(D) of interaction with FIXa, e.g., there may be difference of less than 25%, optionally less than 10%, in the FIXa-binding arm's affinity for FIX compared with the affinity for FIXa. There may be no statistically significant difference in K_(D) of interaction with FIX compared with FIXa.

As described elsewhere herein, affinity may be determined using surface plasmon resonance (SPR), e.g., with the binding arm coupled to a solid surface, optionally as a dimer (e.g., as monospecific IgG), with the antigen in solution as analyte, at 25° C.

FX Binding

An antigen-binding molecule according to the present invention, or a FX-binding polypeptide arm thereof, may bind the EC domain of human FX with a K_(D) of 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. For example, K_(D) may be between 5 μM and 1 nM, e.g., between 5 μM and 10 nM.

The K_(D) may be between 0.1 μM and 2 μM, e.g., between 0.1 μM and 1 μM, e.g., between 0.15 and 0.3 μM. The K_(D) may be 0.6 μM or less, 0.5 μM or less, 0.4 μM or less, 0.3 μM or less, or 0.25 μM or less. The K_(D) may be at least 0.1 μM.

The K_(D) may be 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. The K_(D) may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. The K_(D) may be at least 0.001 nM, for example at least 0.01 nM or at least 0.1 nM. For example, the K_(D) may be between 1-100 nM. K_(D) may be between 1-10 nM.

As described elsewhere herein, affinity may be determined using surface plasmon resonance (SPR), e.g., with the binding arm coupled to a solid surface, optionally as a dimer (e.g., as monospecific IgG), with the antigen in solution as analyte, at 25° C.

Measurement of Antigen-Binding Affinity

The affinity of an antigen-binding molecule for binding FIX, FIXa, FX and FXa may be quantified in terms of the equilibrium dissociation constant K_(D), the ratio Ka/Kd of the association or on-rate (Ka) and the dissociation or off-rate (kd) of the binding interaction. K_(D), Ka and Kd for antigen binding can be measured using surface plasmon resonance (SPR). Example SPR procedure and conditions are set out in Example 10.

Quantification of affinity may be performed using SPR with the antigen-binding polypeptide arm in monovalent form, e.g., antibody Fab or Fv comprising the antigen binding site, or heterodimeric immunoglobulin (e.g., IgG) having a single antigen-binding arm for the antigen in question. Alternatively, it may be convenient to determine affinity for the antigen-binding polypeptide arm in bivalent form, for example IgG comprising homodimeric antigen-binding arms. SPR may comprise coating dimers of the antigen-binding polypeptide arm on to a biosensor chip (directly or indirectly), exposing the antigen-binding polypeptide arms to antigen in buffered solution at a range of concentrations, dectecting binding, and calculating the equilibrium dissociation constant K_(D) for the binding interaction. SPR may be performed at 25° C. A suitable buffered solution is 150 mM NaCl, 0.05% detergent (e.g., P20) and 3 mM EDTA, pH 7.6. HBS-P 1× (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20 pH 7.6) with 2.5 mM CaCl₂. is an example buffer. The binding data can be fitted to a 1:1 model using standard algorithms, which may be inherent to the instrument used. A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc).

Cross-Reactivity

Regulatory bodies may require candidate therapeutic molecules to have demonstrated therapeutic efficacy in laboratory animals before they advance to human clinical trials. An example of an acquired haemophilia A animal model is a cynomolgus monkey that is rendered deficient in blood clotting through administration of a FVIII-neutralising antibody or a small molecule inhibitor against FVIII, thereby replicating the phenotype of a human haemophilia A patient. To enable testing of bispecific antigen-binding molecules in animal models, it is desirable for the binding site of each arm to be cross-reactive with the corresponding antigen from one or more non-human mammals. Thus, the FIXa binding site of the antigen-binding molecule may bind murine (e.g., mouse or rat), rabbit or non-human primate (e.g., cynomolgus monkey) FIXa as well as human FIXa, and the FX binding site may bind murine (e.g., mouse or rat), rabbit or non-human primate (e.g., cynomolgus monkey) FXa as well as human FXa.

One way to quantify the extent of species cross-reactivity of an antigen-binding molecule (or, more precisely, of its antigen binding site) is as the fold-difference in its affinity for antigen or one species compared with antigen of another species, e.g., fold difference in affinity for human antigen vs cynomolgus antigen. Affinity may be quantified as K_(D), referring to the equilibrium dissociation constant of the binding of the antigen to the antigen-binding molecule. K_(D) may be determined by SPR as described elsewhere herein.

A species cross-reactive binding molecule may have a fold-difference in affinity for binding human and non-human antigen that is 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less or 5-fold or less. To put it another way, the K_(D) of binding the extracellular domain of the human antigen may be within 30-fold, 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of the K_(D) of binding the extracellular domain of the non-human antigen.

Preferably, the binding affinities of human and non-human antigen are within a range of 10-fold or less, more preferably within 5-fold or within 2-fold. K_(D) for binding non-human FIXa, e.g., as determined by surface plasmon resonance, may be up to 10-fold (preferably up to 5-fold or up to 2-fold) greater or up to 10-fold lower (preferably up to 5-fold or up to 2-fold lower) than the Kd for binding human FIXa. Similarly, K_(D) for binding non-human FX, e.g., as determined by SPR, may be up to 10-fold (preferably up to 5-fold or up to 2-fold) greater or up to 10-fold (preferably up to 5-fold or up to 2-fold) lower than the Kd for binding human FX. Methods of determining affinity are described elsewhere herein.

Binding molecules can also be considered species cross-reactive if the K_(D) for binding antigen of both species meets a threshold value, e.g., if the K_(D) of binding human antigen and the K_(D) of binding non-human antigen are both 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. The K_(D) may be 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. The K_(D) may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

While species cross-reactivity for binding antigen of different species may be advantageous, selectivity of the FIXa binding arm and the FX binding arm for their respective antigens is nevertheless desirable to avoid unwanted side effects. Thus, within the body, FIX/FIXa and FX/FXa are preferably the only antigens bound by the antigen-binding molecule.

Enhancement of FIXa-Mediated Activation of FX

The ability of a bispecific antigen-binding molecule to enhance the FIXa-mediated activation of FX to FXa may be determined in assays in vitro or in vivo.

A suitable in vitro assay is the FX activation assay exemplified in Example 3 and Example 7 and illustrated in FIG. 7. The assay comprises

-   -   (i) contacting the bispecific antigen-binding molecule with FIXa         and FX under conditions suitable for formation of FXa (e.g., in         the presence of phospholipid, in buffered solution at 37° C.)     -   (ii) adding substrate that is cleavable by FXa to generate a         detectable product, and     -   (iii) detecting, and optionally quantifying, the presence of the         detectable product.

A detailed protocol is set out in Example 7.

The level of product may be compared with a control assay in which FIXa-FX bispecific antigen-binding molecule is absent from the reaction mixture. Significant difference in product level in the assay with the bispecific compared with control indicates that the bispecific is able to enhance FIXa-mediated activation of FX. FVIII may be included as a positive control.

The level of product may be compared with an assay in which the FIXa-FX bispecific antigen-binding molecule is emicizumab. A bispecific according to the present invention may enhance the FIXa-mediated activation of FX to FXa to the same or similar extent (e.g., within 10% difference or within 5% difference) as emicizumab, or to a greater extent (e.g., more than 10% more activation of FX to FXa than is achieved with emicizumab as measured by the level of detectable product). Preferably the bispecific antibody enhances the FIXa-mediated activation of FX to FXa to at least the same extent as emicizumab. The assay is typically performed at physiological temperature of 37 degrees C. Suitable concentrations of bispecific for use in the assay are indicated in the Examples herein, e.g., 12.5 μg/ml (10.4 nM) or 125 nM.

Another suitable assay is to measure the activated partial thromboplastin time (aPTT) in FVIII-deficient plasma, which may be performed in the presence or the absence of inhibitors and can be used to compare the activity of bispecific molecules with recombinant human FVIII. This assay is exemplified in Example 8. aPTT is an end point assay which provides a global overview of blood clot formation and provides coagulation time as the assay read-out. FVIII-deficient plasma would typically have a coagulation time of around 80-90 seconds in the aPTT assay. Bispecific antigen binding molecules of the present invention are effective to reduce the coagulation time in an aPTT assay (compared with a negative control). The coagulation time of human FVIII-deficient in an aPTT assay with a bispecific antigen binding molecule according to the present invention may for example be the same as or less than that of the coagulation time with recombinant human FVIIIa. Physiological clotting time for normal (FVIII+) human plasma is typically <40 seconds, e.g., in the range of 37-34 s. Similar values are achievable with FVIII-deficient plasma upon provision of activated FVIIIa, which provides a convenient way of standardising the assay through calibration of the apparatus/measurement against reference values. Alternatively, coagulation time of normal (FVIII+) human plasma may be used for reference, the aPTT assay being begun by induction of coagulation through the addition of calcium. The assay is typically performed at physiological temperature of 37 degrees C. Suitable concentrations of bispecific for use in the assay are indicated in the Examples herein, and include 0.1 mg/ml (44 nM), 0.3 mg/ml (133 nM) and 0.5 mg/ml (222 nM).

A bispecific antigen-binding molecule of the present invention may give a coagulation time in the aPTT assay of within 10 seconds of that of FVIIIa (i.e., up to 10 seconds more than or up to 10 seconds less than the coagulation time of the aPTT assay with FVIIIa). Preferably, the coagulation time in the aPTT assay with a bispecific antigen binding molecule of the invention is less than that with FVIIIa. The bispecific antigen-binding molecule may reduce the coagulation time to less than 40 seconds, less than 35 seconds, or less than 30 seconds. The coagulation time may be between 20 and 40 seconds, e.g., between 20 and 30 seconds. Preferably the coagulation time is 22-28 seconds, e.g., 24-26 seconds.

Another measure of function is the rate at which thrombin is generated in FVIII-deficient blood plasma in the presence of the bispecific antigen-binding molecule. Activity of a bispecific antibody may be measured in a thrombin generation assay (TGA) [10]. A number of thrombin generation assays have been described, as recently reviewed [11]. Essentially, a TGA comprises measuring the conversion (activation) of prothrombin to thrombin over time following addition of a test molecule (here, the candidate bispecific antibody), where thrombin is detected via its cleavage of a substrate to form a detectable product.

With reference to FIG. 1, it will be remembered that the extrinsic tissue factor (TF) pathway exists to initiate the coagulation cascade. Cells expressing TF normally reside outside of the vasculature, and upon tissue damage such TF-bearing cells come into contact with circulating platelets. TF acts as a co-factor to facilitate the activation of small amounts of factors IX and X by factor Vila. Activated factor Xa and factor V form a prothrombinase complex on TF-bearing cells, generating a limited amount of thrombin. The newly generated thrombin activates platelets which have accumulated at the site of injury and factor XI which is present on the platelets. Platelet bound FXIa is required to ensure further activation of FIXa. Given the TGA is an ex vivo assay, TF-bearing cells are absent and a coagulation trigger must be supplied to initiate the cascade. Commercial assays typically use a recombinant TF/phospholipid mixture to initiate coagulation [11]. The TGA method exemplified herein (see Example 13) uses a factor IXa/phospholipid mixture as the trigger, although other upstream activators such as FXIa could be used.

To perform the TGA, FVIII-deficient plasma is contacted with (i) the trigger reagent, (ii) a substrate convertable by thrombin to a detectable product, e.g., a fluorogenic or chromogenic substrate which produces a visually detectable product on cleavage by thrombin, and (iii) the test molecule (e.g., bispecific antibody), to create conditions under which the presence of FVIII-mimetic activity would result in thrombin generation and hence a signal from the detectable product. Typically, the plasma will lack free metal ions such as calcium, which are required in the blood clotting cascade (FIG. 1). Ca²⁺ ions may be supplied (e.g., as CaCl₂ in solution) to initiate the assay, e.g., it may be contained within the substrate solution. Following initiation, generation of the detectable product (representing generation of thrombin) is monitored (preferably continuously, or at frequent intervals, e.g., about every 20 seconds) over time, e.g., by detecting fluorescence or colour. A plate reader may be used, e.g., to monitor conversion of a fluorogenic substrate into a fluorophore. TGA is performed at physiological temperature of 37 degrees C.

Fluorescence may be converted to thrombin concentration by calibrating against known concentrations of thrombin added to control plasma. A thrombogram may then be generated (FIG. 26, FIG. 27).

Preferably, bispecific antibodies (or other test molecules) are suitably purified for use in the TGA (e.g., by protein A chromatography and ion exchange chromatography or hydrophobic interaction chromatography), e.g., to provide the bispecific in a composition of at least 95% bispecific heterodimer (i.e., no more than 5% homodimeric or other antibody contaminants should be present). Preferably the test molecule is provided as close to 100% purity as possible. It may be about 98, 99% or 100% pure bispecific.

Approximate reference ranges for plasma from healthy individuals in a fluorogenic TGA are Cmax 200 to 450 nM and Tmax 5 to 8 minutes [11]. Activity in a TGA can also be compared against published representative thrombin generation curves for plasma from healthy individuals, patients with severe FVIII deficiency and patients with severe FVIII deficiency after FVIII infusion [12]. For standardisation, performance in the TGA may also be compared against a calibrator which represents a positive control molecule at known concentration. A dilution series of the test bispecific may be compared against the calibrator at a series of known fixed concentrations. A suitable calibrator is an emicizumab calibrator. Emicizumab calibrator is available commercially, prepared from FVIII immunodepleted citrated human plasma spiked with 100 μg/mL emicizumab (Hemlibra®) and further comprising buffer and stabilisers. It is supplied in lyophilised form and is reconstituted in water before use in the TGA. The exact concentration of emicizumab in the calibrator phial is known, so the activity of a test bispecific molecule in the assay can be compared against the activity of the calibrator after normalising for concentration. As an alternative control for comparison of a bispecific antibody against emicizumab, performance of the test bispecific antibody in the TGA may be compared against performance of a control bispecific antibody having the amino acid sequence of emicizumab, wherein the test bispecific antibody and the bispecific antibody having the amino acid sequence of emicizumab are tested under identical conditions in the TGA.

The TGA may be used to characterise six aspects of thrombin generation: lag time (lag), time to peak (Tmax), maximal peak height (Cmax), endogenous thrombin potential (ETP), velocity index (VI) and the “tail start” or return to baseline. The lag time represents the initiation phase before the thrombin peak begins to be generated, where addition of a trigger results in the activation of the coagulation cascade. Once initiated, large amounts of thrombin are quickly generated during the propagation phase. The time to peak represents the time taken (Tmax) to reach maximal thrombin peak height (Cmax), the ETP represents the total amount of thrombin generated and the velocity index characterises the slope between the lag time and the time to peak. The return to baseline (tail start) reflects the inhibition (by activated protein C) of thrombin formation and the inactivation (by antithrombin) of thrombin already formed. The Cmax and/or Tmax is typically the key measure used to represent activity in the TGA. References values in the TGA (e.g., Cmax, Tmax, lagtime etc.) may be determined for the bispecific at a fixed concentration, e.g, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM or 300 nM. Parameters may be measured a series of concentrations, e.g., at 1 nM, 3 nM, 10 nM, 30 nM, 100 nM and 300 nM and/or other concentrations to obtain a complete dose response curve, allowing EC50 values to then be determined. The dose response curve can be fitted using a non-linear log(antibody) vs response variable slope model (e.g., variable slope 4 parameter logistic regression model, which may be performed using GraphPad Prism v8.0.0). EC50 is the concentration of test molecule (e.g., antibody) at which half-maximal effect is reached (half way between baseline and maximal value of the measured parameter). EC50 can be determined from the dose response curve. Worked examples with EC50 data are presented in Example 14 herein.

In one embodiment, the TGA comprises:

(a) contacting FVIII-deficient plasma lacking free calcium ions with

(i) a trigger reagent comprising a factor IXa/phospholipid mixture,

(ii) a solution comprising a fluorogenic substrate (e.g., 2 nM ZGGR-AMC fluorogenic substrate) which produces a visually detectable fluorophore on cleavage by thrombin, and calcium ions (e.g., 100 mM CaCl₂)), (for example, the FluCa reagent from Stago), and

(iii) the bispecific antibody (e.g., at a purity of 95%-100%, e.g., 99%-100%),

(b) incubating the plasma at 37 degrees C. under conditions in which the presence of FVIII-mimetic activity would result in thrombin generation and hence a signal from the fluorophore, (c) detecting fluorescence over time to monitor conversion of the fluorogenic substrate to fluorophore, (d) calibrating detected fluorescence against fluorescence from solutions of thrombin at predetermined concentrations, and (e) determining one or more parameters of a thrombogram, wherein said parameters are:

maximal thrombin peak height (Cmax) that is reached,

time taken (Tmax) to reach maximal thrombin peak height, and/or

length of the initiation phase before the thrombin peak begins to be generated (lag time).

Said one or more parameters are determined at a series of concentrations of the bispecific antibody to obtain a complete dose response curve including baseline and top plateau (maximal value) of response. A dose response curves may be fitted to the data points using a non-linear log[antibody] vs response parameter variable slope model (4 parameter logistic regression model). EC50 is determined from said dose response curve. Said one or more parameters, or EC50 for said one or more parameters, may be compared between the test bispecific antibody and emicizumab (e.g., emicizumab calibrator, as available from Enzyme Research Laboratories).

A bispecific antibody according to the present invention preferably exhibits a potency that is similar to or greater than that of emicizumab in a fluorimetric TGA. Higher potency may be represented by lower EC50 for one or more parameters in said assay, e.g., Cmax, Tmax or lagtime. As demonstrated in the Examples herein, embodiments of the present invention consistently demonstrated greater potency than emicizumab at lower concentrations. See, for example, the results presented in FIG. 29, FIG. 30, FIG. 31, FIG. 33 and FIG. 34.

The maximal response (e.g., highest Cmax, lowest Tmax, shortest lagtime, etc) in the fluorimetric TGA is also noteworthy. Maximal response is the level at which the measured parameter (e.g., Cmax) plateaus with increasing antibody concentration, and represents the maximum achievable level (e.g., the maximal Cmax). An excessive maximal response may be associated with increased risks of overdosing the bispecific molecule, including risk of consumption coagulopathy or disseminated intravascular coagulation (DIC) which is characterised by abnormally increased activation of procoagulant pathways. Hypercoagulability may compromise patient safety through coagulopathy events such as arterial/venous thrombosis, embolism and thrombotic microangiopathy, and would thus narrow the therapeutic window, i.e., the range of dose or plasma concentration at which a beneficial effect is achieved without unacceptable side effects or risk of adverse events.

Since emicizumab has received regulatory approval based on a safety profile deemed acceptable in human clinical trials, the maximal response of emicizumab in the TGA represent established safe limits. Optionally, bispecifics of the present invention have a maximal Cmax and/or maximal Tmax response in the TGA which is not more than 20% (e.g., not more than 15% or not more than 10%) different from that of emicizumab. These reference values may be determined using an emicizumab calibrator or a sequence identical analogue of emicizumab.

Bispecifics of the present invention may demonstrate maximal responses in the TGA as follows:

-   -   Cmax in the TGA not exceeding 500 nM. Optionally the maximal         response for Cmax does not exceed 450 nM, e.g., does not exceed         400 nM. Maximal response for Cmax may be between 200 and 450 nM,         e.g., between 250 and 350 nM; and/or     -   Tmax in the TGA not lower than a maximal response of 1 minute.         Optionally the maximal response for Tmax is not less than 5         minutes, not less than 4 minutes, not less than 3 minutes or not         less than 2 minutes. Maximal response for Tmax may be between 2         and 10 minutes, e.g., between 2 and 8 minutes or between 5 and 8         minutes.

A bispecific antigen-binding molecule according to the present invention may have a Cmax in the range of 100 to 450 nM (e.g., 200 to 450 nM) as determined by fluorimetric TGA, e.g., wherein the bispecific antibody is at a concentration of 100 nM or 300 nM in said assay. The Cmax is preferably at least 200 nM, more preferably at least 250 nM or at least 300 nM. The Cmax of the bispecific may be the same or similar to (e.g., within 10% difference from) the Cmax of emicizumab, or it may be greater than that of emicizumab. The bispecific may have a Cmax EC50 in said assay that is within 10% of the Cmax EC50 of emicizumab, or that is lower. Where the EC50 is lower than that of emicizumab, there may be at least a 2-fold, at least a 3-fold, at least a 4-fold or at least a 5-fold difference in Cmax EC50 in the TGA between the bispecific of the present invention and emicizumab. Optionally the Cmax EC50 in the TGA may be up to 10-fold, up to 15-fold or up to 20-fold different.EC50 of the Cmax for the bispecific antigen-binding molecule in the fluorimetric TGA may be less than 50 nM, e.g., between 1 nM and 50 nM, between 5 nM and 20 nM, or between 5 nM and 10 nM.

A bispecific antigen-binding molecule according to the present invention may have a Tmax of 8 minutes or under, e.g., in the range of 4 to 8 minutes, as determined by fluorimetric TGA, e.g., wherein the bispecific antibody is at a concentration of 100 nM or 300 nM in said assay. The Tmax of the bispecific may be the same or similar to (e.g., within 10% difference from) the Tmax of emicizumab, or it may be less than that of emicizumab. The bispecific may have a Tmax EC50 in said assay that is within 10% of the Tmax EC50 of emicizumab, or that is lower.

EC50 of the Tmax for the bispecific antigen-binding molecule in the fluorimetric TGA may be less than 5 nM, e.g., less than 3 nM or less than 2 nM. It may be between 1 nM and 5 nM, e.g., between 1 nM and 2 nM.

A bispecific antigen-binding molecule according to the present invention may have a lag time of 2-6 minutes as determined by fluorometric TGA, e.g., wherein the bispecific antibody is at a concentration of 100 nM or 300 nM in said assay. The lagtime of the bispecific may be the same or similar to (e.g., within 10% difference from) the lagtime of emicizumab, or it may be lower than that of emicizumab. The bispecific may have a lagtime EC50 in said assay that is within 10% of the lagtime EC50 of emicizumab, or that is lower.

Bispecific Antigen-Binding Molecules

The bispecific antigen-binding molecule comprises a FIXa binding polypeptide arm and a FX binding polypeptide arm. It may be a multi-chain or single-chain polypeptide molecule. While the FIXa binding polypeptide arm and the FX binding polypeptide arm represent different moieties of the bispecific molecule, one polypeptide can optionally form all or part of both the FIXa binding arm and the FX binding arm.

A polypeptide binding arm is the region of the bispecific molecule that comprises the binding site for one of the antigens (FIXa or FX). One or both antigen-binding sites of a bispecific molecule can be provided by a set of complementarity determining regions (or peptide loops) in a polypeptide arm, wherein the polypeptide arm is any suitable scaffold polypeptide whether that of an antibody (e.g., an antibody Fv region) or a non-antibody molecule. A binding arm may comprise one or more than one (e.g., two) polypeptides or parts (e.g., domains) thereof.

The invention is described in detail herein with reference to bispecific antibodies, wherein at least one of the antigen binding polypeptide arms is provided by a set of CDRs in an antibody VH and/or VL domain, optionally an Fv region.

Antibodies are immunoglobulins or molecules comprising immunoglobulin domains. Antibodies may be IgG, IgM, IgA, IgD or IgE molecules or molecules including antigen-specific antibody fragments thereof. The term “antibody” covers any polypeptide or protein comprising an antibody antigen-binding site. An antibody antigen-binding site (paratope) is the part of an antibody that binds to and is complementary to the epitope of its target antigen. The term “epitope” refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulphonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

An antibody antigen-binding site is provided by a set of complementarity determining regions (CDRs) in an antibody VH and/or VL domain, and is capable of binding the antigen. In an example, the antibody binding site is provided by a single variable domain, e.g., a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site is provided by a VH/VL pair (an Fv) or two or more such pairs.

The antibody variable domains are the portions of the light and heavy chains of antibodies that include amino acid sequences of complementarity determining regions (CDRs; ie., CDR1, CDR2, and CDR3), and framework regions (FRs). Thus, within each of the VH and VL domains are CDRs and FRs. A VH domain comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Amino acid positions assigned to CDRs and FRs may be defined according to IMGT nomenclature. An antibody may comprise an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework. Example sequences of antibody VH and VL domains and CDRs form part of the present disclosure. The CDRs are defined according to the IMGT system [13]. All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the invention. As described herein, a “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs” includes HCDRs and LCDRs.

An antibody may comprise one or more CDRs, e.g. a set of CDRs, within an antibody framework. The framework regions may be of human germline gene segment sequences. Thus, the antibody may be a human antibody having a VH domain comprising a set of HCDRs in a human germline framework. Normally the antibody also has a VL domain comprising a set of LCDRs, e.g. in a human germline framework. An antibody “gene segment”, e.g., a VH gene segment, D gene segment, or JH gene segment refers to oligonucleotide having a nucleic acid sequence from which that portion of an antibody is derived, e.g., a VH gene segment is an oligonucleotide comprising a nucleic acid sequence that corresponds to a polypeptide VH domain from FR1 to part of CDR3. Human v, d and j gene segments recombine to generate the VH domain, and human v and j segments recombine to generate the VL domain. The D domain or region refers to the diversity domain or region of an antibody chain. J domain or region refers to the joining domain or region of an antibody chain. Somatic hypermutation may result in an antibody VH or VL domain having framework regions that do not exactly match or align with the corresponding gene segments, but sequence alignment can be used to identify the closest gene segments and thus identify from which particular combination of gene segments a particular VH or VL domain is derived. When aligning antibody sequences with gene segments, the antibody amino acid sequence may be aligned with the amino acid sequence encoded by the gene segment, or the antibody nucleotide sequence may be aligned directly with the nucleotide sequence of the gene segment. Germline gene segments corresponding to framework regions of example antibodies described herein are indicated in Table S-12.

An antibody may be a whole immunoglobulin, including constant regions, or may be an antibody fragment. An antibody fragment is a portion of an intact antibody, for example comprising the antigen binding and/or variable region of the intact antibody. The antibody fragment may include one or more constant region domains.

An antibody of the invention may be a human antibody or a chimaeric antibody comprising human variable regions and non-human (e.g., mouse) constant regions. The antibody of the invention for example has human variable regions, and optionally also has human constant regions.

Thus, antibodies optionally include constant regions or parts thereof, e.g., human antibody constant regions or parts thereof, such as a human IgG4 constant region. For example, a VL domain may be attached at its C-terminal end to antibody light chain kappa or lambda constant domains. Similarly, an antibody VH domain may be attached at its C-terminal end to all or part (e.g. a CH1 domain or Fc region) of an immunoglobulin heavy chain constant region derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, such as IgG1 or IgG4.

Digestion of whole (bivalent) immunoglobulins with the enzyme papain results in two identical (monovalent) antigen-binding fragments known as “Fab” fragments, and an “Fc” fragment. The Fc has no antigen-binding activity but has the ability to crystallize. “Fab” when used herein refers to a fragment of an antibody that includes one constant and one variable domain of each of the heavy and light chains. The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The “Fc fragment” refers to the carboxy-terminal portions of both H chains held together by disulphides.

Digestion of antibodies with the enzyme pepsin results in a bivalent F(ab′)2 fragment in which the two arms of the antibody molecule remain linked. The F(ab′)2 fragment is a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single-chain antibodies (e.g., scFv) are another fragment. Two different monovalent monospecific antibody fragments such as scFv may be linked together to form a bivalent bispecific antibody.

“Fv” when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognise and bind antigen, although usually at a lower affinity than the entire binding site.

Preferably, the bispecific antibody is a dual binding antibody, i.e., a bispecific antibody in which both antigen binding domains are formed by a VH/VL pair. Dual binding antibodies include FIT-Ig (see WO2015/103072, incorporated herein by reference), mAb-dAb, dock and lock, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, Kλ-body, orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triple body, Miniantibody, minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-holes, knobs-in-holes with common light chain, knobs-in-holes with common light chain and charge pairs, charge pairs, charge pairs with common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv and scFv4-Ig.

In one embodiment, the bispecific antibody is a bispecific IgG comprising a FIXa-binding polypeptide arm and a FX-binding polypeptide arm, each polypeptide arm comprising a heavy chain and a light chain. The IgG is a tetrameric immunoglobulin comprising

a first pair of antibody heavy and light chains (heavy-light chain pair) comprising a FIXa binding Fv region,

a second heavy-light chain pair comprising a FX binding Fv region,

wherein each heavy chain comprises a VH domain and a constant region, and each light chain comprises a VL domain and a constant region, and wherein the first and second heavy-light chain pairs associate through heterodimerisation of their heavy chain constant regions to form the immunoglobulin tetramer.

Optionally, the two polypeptide arms comprise a common light chain, so the light chain of the first and second heavy-light chain pairs has an identical amino acid sequence (FIG. 5). Alternatively the two polypeptide arms may comprise different light chains.

Bispecific antibody may be monovalent for binding FIXa and for binding FX.

Antibody Constant Regions

As discussed above, antibodies can be provided in various isotypes and with different constant regions. The Fc region of antibodies is recognised by Fc receptors and determines the ability of the antibody to mediate cellular effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement dependent cytotoxicity (CDC) activity and antibody-dependent cell phagocytosis (ADCP) activity. These cellular effector functions involve recruitment of cells bearing Fc receptors to the site of the target cells, resulting in killing of the antibody-bound cell.

In the context of the present invention it is desirable to avoid cellular effector functions such as ADCC, ADCP and/or CDC. Therefore, bispecific antigen-binding molecules according to the present invention may lack Fc effector function, for example they may contain Fc regions that do not mediate ADCC, ADCP and/or CDC, or they may lack Fc regions or lack antibody constant regions entirely. An antibody may have a constant region which is effector null.

An antibody may have a heavy chain constant region that binds one or more types of Fc receptor but does not induce cellular effector functions, i.e., does not mediate ADCC, CDC or ADCP activity. Such a constant region may be unable to bind the particular Fc receptor(s) responsible for triggering ADCC, CDC or ADCP activity.

An antibody may have a heavy chain constant region that does not bind Fcγ receptors, for example the constant region may comprise a Leu235Glu mutation (i.e., where the wild type leucine residue is mutated to a glutamic acid residue), which may be referred to as an “E” mutation, e.g., IgG4-E. Another optional mutation for a heavy chain constant region is Ser228Pro (“P” mutation), which increases stability by reducing Fab arm exchange. A heavy chain constant region may be an IgG4 comprising both the Leu235Glu mutation and the Ser228Pro mutation (EU numbering). This “IgG4-PE” heavy chain constant region is effector null. An alternative effector null human constant region is a disabled IgG1.

Antibody constant regions may be engineered to have an extended half life in vivo. Examples include “YTE” mutations and other half-life extending mutations (Dall'Acqua, Kiener & Wu, JBC 281(33):23514-23524 2006 and WO02/060919, incorporated by reference herein). The triple mutation YTE is a substitution of 3 amino acids in the IgG CH2 domain, these mutations providing tyrosine at residue 252, threonine at residue 254 and glutamic acid at residue 256, numbered according to the EU index of Kabat. As described in the referenced publications, the YTE modification increases the half-life of the antibody compared with the half-life of a corresponding antibody having a human CH2 wild type domain. To provide an increased duration of efficacy in vivo, antibodies of the present invention may include antibody constant regions (e.g., IgG constant regions, e.g., IgG CH2 domains) that have one or more mutations that increase the half life of the antibody compared with the corresponding wild type human constant region (e.g., IgG, e.g., IgG CH2 domain). Half-life may be determined by standard methods, such as are described in WO02/060919.

In some embodiments, a gamma-carboxyglutamic acid-rich (Gla) domain or other membrane-binding domain is included in the bispecific antibody (e.g., at the C terminus of the Fc), to promote localisation of the antibody to the phospholipid membrane at the platelet surface (via interaction between the Gla domain and the membrane), thereby increasing the local concentration of bispecific antibody where FIX and FX are naturally present in vivo. WO2018/145125 described a FVIII mimetic protein comprising a FIX/FX bispecific antibody and a membrane binding domain, e.g., a platelet binding domain such as a C1, C2 domain, a PH domain, a GLA domain or a membrane binding domain of a platelet membrane glycoprotein. As described therein, the membrane-binding domain may be linked to the C terminal of one or both of the heavy chain constant domains of the bispecific antibody. Bispecific antigen binding molecules of the present invention may optionally include the features and molecular formats described in WO2018/145125.

As discussed below, in bispecific IgG formats or other antibody formats where the different antigen binding arms are heterodimerised via constant regions, the constant regions may be engineered to promote heterodimer formation over homodimer formation and/or to facilitate purification of heterodimers from a mixture of different species.

The anti-FIXaxFX bispecific antibody emicizumab contains a heavy chain constant region which includes features designed to promote its assembly, purification and/or therapeutic performance. A bispecific antibody according to the present invention may comprise any one or more of these features. Thus it may comprise a human IgG4 (e.g., IgHG4*03) heavy chain constant region amino acid sequence comprising one or more of the following changes (EU numbering):

Lys196Gln in CH1;

Ser228Pro in the hinge region (P mutation);

Phe296Tyr in the DE turn of CH2;

Glu356Lys in CH3;

Lys439Glu in CH3;

Leu445Pro in CH3;

Deletion of Gly446;

Deletion of Lys447.

One each of the mutations Glu356Lys and Lys439Glu are included in the two oppositely paired heavy chain constant regions within the Fc of the heterodimeric bispecific, i.e., one heavy chain constant region comprises Glu356 and Lys439Glu and the other heavy chain constant region comprises Glu356Lys and Lys439 (see the discussion on charge pairing below).

A bispecific antibody according to the present invention may comprise an Fc region that has any one or more of the features that are present in the Fc region of emicizumab. It may comprise the Fc region of emicizumab. In one embodiment, the amino acid sequences of the heavy chain constant regions are the amino acid sequences of the emicizumab heavy chain constant regions.

Example amino acid sequences for heavy chain constant regions are shown in Table S-100.

Engineering of Bispecific Antibodies to Facilitate Heterodimer Formation and/or Purification

One of the difficulties with using bispecific antibodies in the clinic has historically been the difficulty of producing them in large quantities and at pharmaceutical grade purity. The “traditional” bispecific IgG format comprises two different pairs of heavy and light chains, thus 4 different polypeptide chains, which if expressed together could assemble into 10 different potential antibody molecules. The mixture of species will include homodimers (homodimeric anti-FIXa binding arms and homodimeric anti-FX binding arms), molecules in which one or both light chains are swapped between the H-L pairs, as well as the “correct” bispecific heterodimeric structure.

Alternative molecular formats have been developed which avoid this potential mis-pairing, and several examples are provided herein. These include F(ab′)2, e.g., prepared by chemical coupling or leucine zipper (fos:jun) assembly, diabodies, and scFv heterodimers. Nevertheless, it remains desirable to be able to use bispecific IgG, to reflect the native structure of antibodies in the bloodstream and to minimise immunogenicity of the administered therapeutic molecule. Additionally, a full length bispecific antibody may have a longer serum half-life.

“Knobs into holes” technology for making bispecific antibodies was described in [14] and in U.S. Pat. No. 5,731,168, both incorporated herein by reference. The principle is to engineer paired CH3 domains of heterodimeric heavy chains so that one CH3 domain contains a “knob” and the other CH3 domains contains a “hole” at a sterically opposite position. Knobs are created by replacing small amino acid side chain at the interface between the CH3 domains, while holes are created by replacing large side chains with smaller ones. The knob is designed to insert into the hole, to favour heterodimerisation of the different CH3 domains while destabilising homodimer formation. In in a mixture of antibody heavy and light chains that assemble to form a bispecific antibody, the proportion of IgG molecules having paired heterodimeric heavy chains is thus increased, raising yield and recovery of the active molecule

Mutations Y349C and/or T366W may be included to form “knobs” in an IgG CH3 domain. Mutations E356C, T366S, L368A and/or Y407V may be included to form “holes” in an IgG CH3 domain. Knobs and holes may be introduced into any human IgG CH3 domain, e.g., an IgG1, IgG2, IgG3 or IgG4 CH3 domain. A preferred example is IgG4. As noted, the IgG4 may include further modifications such as the “P” and/or “E” mutations. An example IgG4-PE sequence and other example constant regions including knobs-into-holes mutations are shown in Table S-100. The IgG4 type a (“ra”) sequence contains substitutions Y349C and T366W (“knobs”), and the IgG4 type b (“yb”) sequence contains substitutions E356C, T366S, L368A, and Y407V (“holes”). Both ra and yb also contain the “P” substitution at position 228 in the hinge (S228P), to stabilise the hinge region of the heavy chain. Both ra and yb also contain the “E” substitution in the CH2 region at position 235 (L235S), to abolish binding to FcγR. Thus the relevant sequence of the IgG4-PE heavy chain is ppcpPcpapefEggps (SEQ ID NO: 401). A bispecific antigen binding molecule of the present invention may contain an IgG4 PE human heavy chain constant region (e.g., SEQ ID NO: 143), optionally two such paired constant regions, optionally wherein one has “knobs” mutations and one has “holes” mutations, e.g., wherein one heavy chain constant region has a sequence SEQ ID NO: 144 (knobs) and one heavy chain constant region has a sequence SEQ ID NO: 145 (holes).

A further advance in bispecific IgG engineering was the idea of using a common light chain, as described in WO98/50431. Bispecific antibodies comprising two heavy-light chain pairs were described, in which the variable light chains of both heavy-light chain pairs had a common sequence. WO98/50431 described combining the common light chain approach with specific complementary interactions in the heavy chain heterodimerisation interface (such as knobs-into-holes) to promote heterodimer formation and hinder homodimer formation. In combination, these approaches enhance formation of the desired heterodimer relative to undesired heterodimers and homodimers.

While knobs-into-holes technology involves engineering amino acid side chains to create complementary molecular shapes at the interface of the paired CH3 domains in the bispecific heterodimer, another way to promote heterodimer formation and hinder homodimer formation is to engineer the amino acid side chains to have opposite charges. Association of CH3 domains in the heavy chain heterodimers is favoured by the pairing of oppositely charged residues, while paired positive charges or paired negative charges would make homodimer formation less energetically favourable. WO2006/106905 described a method for producing a heteromultimer composed of more than one type of polypeptide (such as a heterodimer of two different antibody heavy chains) comprising a substitution in an amino acid residue forming an interface between said polypeptides such that heteromultimer association will be regulated, the method comprising:

(a) modifying a nucleic acid encoding an amino acid residue forming the interface between polypeptides from the original nucleic acid, such that the association between polypeptides forming one or more multimers will be inhibited in a heteromultimer that may form two or more types of multimers;

(b) culturing host cells such that a nucleic acid sequence modified by step (a) is expressed; and

(c) recovering said heteromultimer from the host cell culture,

wherein the modification of step (a) is modifying the original nucleic acid so that one or more amino acid residues are substituted at the interface such that two or more amino acid residues, including the mutated residue(s), forming the interface will carry the same type of positive or negative charge.

An example of this is to suppress association between heavy chains by introducing electrostatic repulsion at the interface of the heavy chain homodimers, for example by modifying amino acid residues that contact each other at the interface of the CH3 domains, including:

positions 356 and 439

positions 357 and 370

positions 399 and 409,

the residue numbering being according to the EU numbering system.

By modifying one or more of these pairs of residues to have like charges (both positive or both negative) in the CH3 domain of a first heavy chain, the pairing of heavy chain homodimers is inhibited by electrostatic repulsion. By engineering the same pairs or pairs of residues in the CH3 domain of a second (different) heavy chain to have an opposite charge compared with the corresponding residues in the first heavy chain, the heterodimeric pairing of the first and second heavy chains is promoted by electrostatic attraction.

Amino acids at the heavy chain constant region CH3 interface were modified to introduce charge pairs, the mutations being listed in Table 1 of WO2006/106905. It was reported that modifying the amino acids at heavy chain positions 356, 357, 370, 399, 409 and 439 to introduce charge-induced molecular repulsion at the CH3 interface had the effect of increasing efficiency of formation of the intended bispecific antibody. For example, one heavy chain constant region may be an IgG4 constant region containing mutation K439E (positively charged Lys replaced by negatively charged Glu) and the other heavy chain constant region may be an IgG4 constant region containing mutation E356K (negatively charged Glu replaced by positively charged Lys), using EU numbering. “Charge pairing” results from spatial proximity of residues 439 and 356 in an Fc region assembled from heterodimerisation of these two constant regions.

Where two different heavy chain constant regions are used, these may be connected to the two different VH domains of the antibody in either orientation. For example,

a first heavy chain may comprise an anti-FIX VH domain and a constant region comprising K439E, and a second heavy chain may comprise an anti-FX VH domain and a constant region comprising E356K, or

a first heavy chain may comprise an anti-FIX VH domain and a constant region comprising E356K, and a second heavy chain may comprise an anti-FX VH domain and a constant region comprising K439E.

WO2006/106905 also exemplified bispecific IgG antibodies binding FX and FIXa in which the CH3 domains of IgG4 were engineered with knobs-into-holes mutations. Type a Type a (IgG4γa) was an IgG4 substituted at Y349C and T366W, and type b (IgG4γb) was an IgG4 substituted at E356C, T366S, L368A, and Y407V.

In another example, introduction of charge pairs in the antibody VH and VL domains was used to inhibit the formation of “incorrect” VH-VL pairs (pairing of VH from one antibody with VL of the other antibody). In one example, Q residues in the VH and VL were changed to K or R (positive), or to E or D (negative), to inhibit hydrogen bonding between the Q side chains and to introduce electrostatic repulsion.

Further examples of charge pairs were disclosed in WO2013/157954, which described a method for producing a heterodimeric CH3 domain-comprising molecule from a single cell, the molecule comprising two CH3 domains capable of forming an interface. The method comprised providing in the cell

(a) a first nucleic acid molecule encoding a first CH3 domain-comprising polypeptide chain, this chain comprising a K residue at position 366 according to the EU numbering system and

(b) a second nucleic acid molecule encoding a second CH3 domain-comprising polypeptide chain, this chain comprising a D residue at position 351 according to the EU numbering system,

the method further comprising the step of culturing the host cell, allowing expression of the two nucleic acid molecules and harvesting the heterodimeric CH3 domain-comprising molecule from the culture.

Further methods of engineering electrostatic interactions in polypeptide chains to promote heterodimer formation over homodimer formation were described in WO2011/143545.

Another example of engineering at the CH3-CH3 interface is strand-exchange engineered domain (SEED) CH3 heterodimers. The CH3 domains are composed of alternating segments of human IgA and IgG CH3 sequences, which form pairs of complementary SEED heterodimers referred to as “SEED-bodies” [15; WO2007/110205].

Bispecifics have also been produced with heterodimerised heavy chains that are differentially modified in the CH3 domain to alter their affinity for binding to a purification reagent such as Protein A. WO2010/151792 described a heterodimeric bispecific antigen-binding protein comprising

a first polypeptide comprising, from N-terminal to C-terminal, a first epitope-binding region that selectively binds a first epitope, an immunoglobulin constant region that comprises a first CH3 region of a human IgG selected from IgG1, IgG2, and IgG4; and

a second polypeptide comprising, from N-terminal to C-terminal, a second epitope-binding region that selectively binds a second epitope, an immunoglobulin constant region that comprises a second CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, wherein the second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 domain to Protein A.

The Fc region may thus comprise one or more mutations to promote differential purification of the active heterodimer from homodimer species. The CH3 of one heavy chain constant region may comprise the mutation His435Arg and/or Tyr436Phe (EU numbering) [16] while the CH3 of the other heavy chain constant region lacks said mutations. Emicizumab, for example, comprises an Fc region in which one CH3 comprises His435 and the other CH3 comprises His435Arg.

The bispecifics of the present invention may employ any of these techniques and molecular formats as desired.

Generating and Modifying Antibodies

Methods for identifying and preparing antibodies are well known. Isolated (optionally mutated) nucleic acid encoding antibodies (or heavy-light chain pairs or polypeptide binding arms thereof) described herein may be introduced into host cells, e.g., CHO cells as discussed. Host cells are then cultured under conditions for expression of the antibody (or of the antibody heavy and/or light chain variable domain, heavy-light chain pair, or polypeptide binding arm) to produce the desired antibody format. Some possible antibody formats are described herein, e.g., whole immunoglobulins, antigen-binding fragments, and other designs.

Variable domain amino acid sequence variants of any of the VH and VL domains or CDRs whose sequences are specifically disclosed herein and may be employed in accordance with the present invention, as discussed.

Alterations to nucleic acid encoding the antibody heavy and/or light chain variable domain may be performed, such as mutation of residues and generation of variants, as described herein. There are many reasons why it may be desirable to create variants, which include optimising the antibody sequence for large-scale manufacturing, facilitating purification, enhancing stability or improving suitability for inclusion in a desired pharmaceutical formulation. Protein engineering work can be performed at one or more target residues in the antibody sequence, e.g., to substituting one amino acid with an alternative amino acid (optionally, generating variants containing all naturally occurring amino acids at this position, with the possible exception of Cys and Met), and monitoring the impact on function and expression to determine the best substitution. It is in some instances undesirable to substitute a residue with Cys or Met, or to introduce these residues into a sequence, as to do so may generate difficulties in manufacturing—for instance through the formation of new intramolecular or intermolecular cysteine-cysteine bonds. Where a lead candidate has been selected and is being optimised for manufacturing and clinical development, it will generally be desirable to change its antigen-binding properties as little as possible, or at least to retain the affinity and potency of the parent molecule. However, variants may also be generated in order to modulate key antibody characteristics such as affinity, cross-reactivity or neutralising potency.

One or more amino acid mutations may optionally be made in framework regions of an antibody VH or VL domain disclosed herein. For example, one or more residues that differ from the corresponding human germline segment sequence may be reverted to germline. Human germline gene segment sequences corresponding to VH and VL domains of example antibodies herein are indicated in Table S-12.

In a bispecific antigen binding molecule, an antigen-binding site may comprise a set of H and/or L CDRs of any of the disclosed anti-FIX or anti-FX antibodies with one or more amino acid mutations within the disclosed set of H and/or L CDRs. The mutation may be an amino acid substitution, deletion or insertion. Thus for example there may be one or more amino acid substitutions within the disclosed set of H and/or L CDRs. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations e.g. substitutions, within the set of H and/or L CDRs. For example, there may be up to 6, 5, 4, 3 or 2 mutations, e.g. substitutions, in HCDR3 and/or there may be up to 6, 5, 4, 3, or 2 mutations, e.g. substitutions, in LCDR3.

An antibody may comprise a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VH domain as shown in the Tables, and/or comprising a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of those antibodies. Algorithms that can be used to calculate % identity of two amino acid sequences include e.g. BLAST, FASTA, or the Smith-Waterman algorithm, e.g. employing default parameters. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue).

Alterations may be made in one or more framework regions and/or one or more CDRs. Variants are optionally provided by CDR mutagenesis. The alterations normally do not result in loss of function, so an antibody comprising a thus-altered amino acid sequence may retain an ability to bind its antigen. It may retain the same quantitative binding ability as an antibody in which the alteration is not made, e.g. as measured in an assay described herein. The antibody comprising a thus-altered amino acid sequence may have an improved ability to bind its antigen.

Alteration may comprise replacing one or more amino acid residue with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 “standard” L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring.

The term “variant” as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, substitutions or additions, yet retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as “conservative”, in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be “non-conservative”, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. In some embodiments amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide).

In some aspects, one can use “synthetic variants”, “recombinant variants”, or “chemically modified” polynucleotide variants or polypeptide variants isolated or generated using methods well known in the art. “Modified variants” can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects include insertion variants, deletion variants or substituted variants with substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. The term “conservative substitution,” when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.). Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered “conservative substitutions” if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.

One can select the amino acid that will substitute an existing amino acid based on the location of the existing amino acid, including its exposure to solvents (i.e., if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of variants.

The invention includes methods of producing polypeptide binding arms containing VH and/or VL domain variants of the antibody VH and/or VL domains shown in the Tables herein. FIXa binding polypeptide arms comprising variant VH domains may be produced by a method comprising

(i) providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent antibody VH domain, an antibody VH domain that is an amino acid sequence variant of the parent antibody VH domain,

wherein the parent antibody VH domain is a VH domain shown in FIG. 20, e.g., N1280H, or is a VH domain comprising the heavy chain complementarity determining regions of any of those VH domains,

(ii) optionally combining the VH domain thus provided with a VL domain, to provide a VH/VL combination, and

(iii) testing the VH domain or VH/VL domain combination thus provided to identify an antibody with one or more desired characteristics.

The VH domain may be any VH domain whose sequence is shown in Table S-9A or FIG. 20, or any VH domain comprising a set of HCDRs (HCDR1, HCDR2 and HCDR3) of a VH domain shown in Table S-9A or FIG. 20.

Desired characteristics of FIXa-binding polypeptide arms, and of bispecific anti-FIXa/FX binding molecules comprising them, are detailed elsewhere herein. For example, the method may comprise confirming that the VH domain or VH/VL domain combination binds FIXa as described herein.

When VL domains are included in the method, the VL domain may be the N0128L VL domain or may be a variant provided by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the N0128L VL domain, or may be a VL domain comprising the light chain complementarity determining regions of the N0128L VL domain. The VL domain may be the 0325L VL domain.

Methods of generating variant antibodies may optionally comprise producing copies of the antibody or VH/VL domain combination. Methods may further comprise producing a bispecific antibody comprising the FIXa binding polypeptide arm, for example by expression of encoding nucleic acid. Suitable methods of expression, including recombinant expression in host cells, are set out in detail herein.

Encoding Nucleic Acids and Methods of Expression

Isolated nucleic acid may be provided, encoding bispecific antigen binding molecules, e.g., bispecific antibodies, according to the present invention. Nucleic acid may be DNA and/or RNA. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode an antibody.

The present invention provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. Exemplary nucleotide sequences are included in the Tables. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

The present invention also provides a recombinant host cell that comprises one or more nucleic acids encoding the antigen binding molecule. Methods of producing the encoded molecule may comprise expression from the nucleic acid, e.g., by culturing recombinant host cells containing the nucleic acid. The bispecific molecule may thus be obtained, and may be isolated and/or purified using any suitable technique, then used as appropriate. A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals.

The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is E. coli. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.

Vectors may contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Nucleic acid encoding an antibody can be introduced into a host cell. Nucleic acid can be introduced to eukaryotic cells by various methods, including calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by expressing the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene, then optionally isolating or purifying the antibody.

Nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques. Nucleic acid encoding a bispecific may be integrated into genomic DNA of a host (e.g., CHO) cell, e.g., into chromosomal DNA, and the resulting recombinant cell may be cultured to express the bispecific. A cell line development process may comprise introducing nucleic acid encoding the bispecific into multiple host cells, and selecting a cell line which expresses a desired level of bispecific antibody (e.g., at least 95% heterodimer, with no more than 5% homodimeric contaminants) at the desired yield (e.g., at least 0.5 g/L or at least 1 g/L). Preferably the cell line will retain stable expression over a number of generations in cell culture, and thus it may maintain these levels of production over a at least 60 generations for example.

The present invention also provides a method that comprises using nucleic acid described herein in an expression system in order to express the bispecific antigen binding molecule. Desirably, the antigen-binding molecules are expressed at a yield of at least 0.5 g/L in the cell supernatant after initial fermentation, preferably at a yield of >2 g/L. Solubility should be >10 mg/ml, preferably >50 mg/ml, without significant aggregation or degradation of the molecules.

To provide medicines suitable for global treatment, antibodies can be produced on a large scale, for instance in cell culture volumes of at least 100 litres or at least 200 litres, e.g., between 100-250 litres. Batch culture, particularly fed-batch culture, is now commonly used for production of biotherapeutics for clinical and commercial use, and such methods may suitably be used in the present invention to generate the antibodies, followed by purification and formulation steps as noted herein. Bioreactors may be metal (e.g., stainless steel) vessels or may be single-use bioreactors.

Formulation and Administration

The bispecific antigen-binding molecules (“bispecifics”) according to the present invention, and their encoding nucleic acid molecules, will usually be provided in isolated form. The bispecifics VH and/or VL domains, and nucleic acids may be provided purified from their natural environment or their production environment. Isolated antigen-binding molecules and isolated nucleic acid will be free or substantially free of material with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are prepared (e.g., cell culture) when such preparation is by recombinant DNA technology in vitro. Optionally an isolated antigen-binding molecule or nucleic acid (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature.

Bispecific antibody may be purified (e.g., from cell culture supernatant) by protein A chromatography and/or ion exchange chromatography. The bispecific antibody may be produced by a method comprising

expressing two antibody heavy chains and common light chain from cultured host cells comprising encoding nucleic acids,

obtaining cell culture comprising the bispecific antibody and monospecific antibodies assembled from the antibody heavy chains and common light chain,

isolating the bispecific antibody and monospecific antibodies from the cell culture (e.g., using protein A chromatography), and

purifying the bispecific antibody from the monospecific antibodies (e.g., using cation exchange chromatography).

Bispecifics or their encoding nucleic acids may be formulated with diluents or adjuvants and still for practical purposes be isolated—for example they may be mixed with carriers if used to coat microtitre plates for use in immunoassays, and may be mixed with pharmaceutically acceptable carriers or diluents when used in therapy. As described elsewhere herein, other active ingredients may also be included in therapeutic preparations. The antigen binding molecules may be glycosylated, either naturally in vivo or by systems of heterologous eukaryotic cells such as CHO cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated. The invention encompasses antibodies having a modified glycosylation pattern.

Typically, an isolated product constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. A bispecific may be substantially free from proteins or polypeptides or other contaminants that are found in its natural or production environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

As discussed elsewhere herein, expression of antibody heavy and light chains for a bispecific antibody may generate unwanted homodimeric species (anti-FIX and anti-FX antibodies) in addition to the active heterodimeric bispecific antibody. Preferably a bispecific is provided in a composition in which the heterodimeric bispecific antibody is represents at least 95% of the total antibody, with homodimeric antibody contaminants being present at 5% or less. The composition may comprise at least 98% or at least 99% heterodimeric bispecific, with homodimeric contaminants representing 0-2% or 0-1% respectively.

The invention provides therapeutic compositions comprising the bispecifics described herein. Therapeutic compositions comprising nucleic acid encoding such bispecifics are also provided. Encoding nucleic acids are described in more detail elsewhere herein and include DNA and RNA, e.g., mRNA. In therapeutic methods described herein, use of nucleic acid encoding the bispecific, and/or of cells containing such nucleic acid, may be used as alternatives (or in addition) to compositions comprising the bispecific molecule itself. Cells containing nucleic acid encoding the bispecific, optionally wherein the nucleic acid is stably integrated into the genome, thus represent medicaments for therapeutic use in a patient. Nucleic acid encoding the bispecific may be introduced into human cells derived from the intended patient and modified ex vivo. Administration of cells containing the encoding nucleic acid to the patient provides a reservoir of cells capable of expressing the bispecific, which may provide therapeutic benefit over a longer term compared with administration of isolated nucleic acid or the isolated bispecific molecule. Nucleic acid encoding the bispecific may be provided for use in gene therapy, comprising introducing the encoding nucleic acid into cells of the patient in vivo, so that the nucleic acid is expressed in the patient's cells and provides a therapeutic effect such as compensating for hereditary factor VIII deficiency.

Compositions may contain suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311. Compositions may comprise the antibody or nucleic acid in combination with medical injection buffer.

Bispecifics, or their encoding nucleic acids, may be formulated for the desired route of administration to a patient, e.g., in liquid (optionally aqueous solution) for injection. An example buffer in which to formulate the bispecific for injection is an aqueous solution of 20 mM sodium acetate, 150 mM arginine hydrochloride, 0.05% w/v polysorbate 80 pH 5.2.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The antigen-binding molecules are preferably administered by subcutaneous injection.

The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. It is envisaged that treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle-free device is also advantageous. With respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the aforesaid antibody may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

The bispecific, nucleic acid, or composition comprising it, may be contained in a medical container such as a phial, syringe, IV container or an injection device. In an example, the bispecific, nucleic acid or composition is in vitro, and may be in a sterile container. In an example, a kit is provided comprising the bispecific, packaging and instructions for use in a therapeutic method as described herein.

One aspect of the invention is a composition comprising a bispecific or nucleic acid of the invention and one or more pharmaceutically acceptable excipients, examples of which are listed above. “Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A pharmaceutically acceptable carrier, excipient, or adjuvant can be administered to a patient, together with a bispecific agent, e.g., any antibody or polypeptide molecule described herein, and does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

In some embodiments, the bispecific will be the sole active ingredient in a composition according to the present invention. Thus, a composition may consist of the antibody or it may consist of the bispecific with one or more pharmaceutically acceptable excipients. However, compositions according to the present invention optionally include one or more additional active ingredients.

Where required (for example, for management of acute bleeds), the bispecific may be combined with one or more other treatments for haemophilia, including recombinant factor VIII (e.g., turoctocog alfa) or recombinant factor Vila (e.g., eptacog alfa). The functional properties and safety profile of bispecifics described herein are believed to be suitable for their safe combination with such further therapeutic agents. The bispecific may be combined with recombinant factor Va (FVa), for example an activated variant FVa as described in U.S. Ser. No. 10/407,488.

Other therapeutic agents that it may be desirable to administer with bispecific or nucleic acids according to the present invention include analgaesic agents. Any such agent or combination of agents may be administered in combination with, or provided in compositions with antibodies or nucleic acids according to the present invention, whether as a combined or separate preparation. The bispecific or nucleic acid according to the present invention may be administered separately and sequentially, or concurrently and optionally as a combined preparation, with another therapeutic agent or agents such as those mentioned.

Multiple compositions can be administered separately or simultaneously. Separate administration refers to the two compositions being administered at different times, e.g. at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One can also administer compositions at 24 hours apart, or even longer apart. Alternatively, two or more compositions can be administered simultaneously, e.g. less than 10 or less than 5 minutes apart. Compositions administered simultaneously can, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components.

Bispecifics, and their encoding nucleic acids, can be used as therapeutic agents. Patients herein are generally mammals, typically humans. A bispecific or nucleic acid may be administered to a mammal, e.g., by any route of administration mentioned herein.

Administration is normally in a “therapeutically effective amount”, this being an amount that produces the desired effect for which it is administered, sufficient to show benefit to a patient. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. A therapeutically effective amount or suitable dose of bispecific or nucleic acid can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known.

Bispecifics may be administered in an amount in one of the following ranges per dose:

about 10 μg/kg body weight to about 100 mg/kg body weight,

about 50 μg/kg body weight to about 5 mg/kg body weight,

about 100 μg/kg body weight to about 10 mg/kg body weight,

about 100 μg/kg body weight to about 20 mg/kg body weight,

about 0.5 mg/kg body weight to about 20 mg/kg body weight, or

about 5 mg/kg body weight or lower, for example less than 4, less than 3, less than 2, or less than 1 mg/kg of the antibody.

The dose of antigen-binding molecule administered may be up to 1 mg/kg. It may be formulated at lower strength for paediatric populations, for example 30-150 mg/mL. The bispecific molecule may be packaged in smaller quantities for a paediatric population, e.g., it may be provided in phials of 25-75 mg, e.g., 30 or 60 mg.

In methods of treatment described herein, one or more doses may be administered. In some cases, a single dose may be effective to achieve a long-term benefit. Thus, the method may comprise administering a single dose of the bispecific, its encoding nucleic acid, or the composition. Alternatively, multiple doses may be administered, usually sequentially and separated by a period of days, weeks or months. Optionally, the bispecific may be administered to a patient once a month, or less frequently, e.g., every two months or every three months.

As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). For treatment to be effective a complete cure is not contemplated. The method can in certain aspects include cure as well. In the context of the invention, treatment may be preventative treatment.

Long half-life is a desirable feature in the bispecifics of the present invention. Extended half-life translates to less frequent administration, with fewer injections being required to maintain a therapeutically effective concentration of the molecule in the bloodstream. The in vivo half life of antigen-binding molecules of the present invention in humans may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or longer. The in vivo half life of antigen-binding molecules in non-human primates such as cynomolgus monkeys may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or longer.

Maintenance of 1% of normal FVIII activity is considered to be a minimum for prophylactic use in haemophilia [1]. In a paper reporting a human clinical trial with ACE910 (emicizumab), in silico population pharmacokinetic modelling and simulations suggested that a weekly dose of 1 mg/kg resulted in a plasma concentration of at least about 300 nM (45 μg/ml), producing a continuous haemostatic effect of at least 10% of normal FVIII activity [4]. This dose was also reported to be well tolerated in patients.

Based on that data, a plasma concentration range of approximately 30 nM (˜4.5 μg/ml) to 300 nM (˜45 μg/ml) would correspond to effective FVIII activity of 1-10%, assuming a linear relationship between antibody concentration and FVIII activity and comparable antibody activity.

Bispecifics according to the present invention exhibit exceptionally high activity at concentrations in the range of 30 nM (˜4.5 μg/ml) to 300 nM (˜45 μg/ml), maintaining strong thrombin generation activity even at relatively low doses. As evidenced by its high potency (see, e.g., Example 14), a bispecific antibody according to the present invention may exhibit therapeutic efficacy at a lower plasma concentration than emicizumab. Therefore it may provide greater therapeutic benefit at an equivalent dose, and it may provide equivalent therapeutic benefit at a lower dose, compared with emicizumab. Patients can thus benefit from smaller and/or less frequent injections, and healthcare providers can benefit from lower associated costs.

The US FDA currently (in guidance issued October 2018) recommends that emicizumab be administered at a loading dose of 3 mg/kg by subcutaneous injection once weekly for the first 4 weeks, followed by a maintenance dose of:

1.5 mg/kg once every week, or

3 mg/kg once every 2 weeks, or

6 mg/kg once every 4 weeks.

Antigen-binding molecules according to the present invention may be provided for administration at regular intervals of one week, two weeks, three weeks, four weeks, or one month.

In a preferred embodiment, the bispecific is administered by subcutaneous injection.

Therapeutic Use

The bispecific antigen-binding molecules of the present invention may be used in a method of treatment of the human or animal body by therapy. Therapeutic indications for the molecules include:

use to treat haemophilia A,

use to treat hereditary factor VIII deficiency,

use to significantly decrease the number of bleeding incidents in haemophilia A patients,

use to substitute for factor VIII function,

and/or

use to promote blood coagulation.

Patients are typically human patients. The patient may be a human diagnosed with haemophilia A or hereditary factor VIII deficiency, or a human who has lower (or absent) factor VIII expression or activity compared with wild type. The patient may be a paediatric patient (e.g., from 2 to less than 18 years of age) or may be an adult. The patient may be a human male. The patient may or may not have inhibitors to factor VIII.

A bispecific molecule of the present invention, or a composition comprising such a bispecific molecule or its encoding nucleic acid, may be used or provided for use in any such method. Use of the bispecific molecule, or of a composition comprising it or its encoding nucleic acid, for the manufacture of a medicament for use in any such method is also envisaged. The method typically comprises administering the antibody or composition to a mammal, e.g., a human patient. Suitable formulations and methods of administration are described elsewhere herein.

There is a presently unmet need for treatment of haemophilia A patients who develop inhibitory allo-antibodies to FVIII. Antigen-binding molecules of the present invention are suitable for use in such patients. Accordingly, in some aspects, a patient treated with a bispecific antigen binding molecule according to the present invention may be resistant to treatment with FVIII owing to the presence of inhibitory antibodies in the bloodstream. Resistance to treatment can be manifested in a reduction of efficacy of the therapy. Such resistance may be detected in in vitro assays (e.g. aPTT assay) with a blood plasma sample from the patient, wherein the therapeutic molecule does not reduce coagulation time to the same level as in an assay with control FVIII-deficient plasma (the latter lacking inhibitory antibodies to the therapeutic molecule).

Patients receiving other treatments for haemophilia, such as bispecific antibodies to FIXa and FX, may also develop inhibitory antibodies to those therapeutic antibodies. As noted, use of human antibodies such as those of the present invention should minimise the risk of this, but inhibitory antibodies may nevertheless be generated in some patients who receive antigen binding molecules of the present invention or other bispecific antigen binding molecules to FIXa and FX. A patient treated with a bispecific antigen binding molecule according to the present invention may be resistant to treatment to a different bispecific antigen binding molecule for FIXa and FX owing to the presence of inhibitory antibodies in the bloodstream. The patient may be resistant to treatment with emicizumab.

Since inhibitory antibodies may be generated through long term therapeutic administration of a drug product, it may be beneficial for patients to alternate or cycle between multiple different treatments, to reduce the risk of their developing inhibitory antibodies. Thus, a bispecific antigen binding molecule of the present invention may be administered to a patient who has previously received treatment with a different FVIIIa-activity replacing polypeptide drug, e.g., a bispecific antigen binding molecule for FIXa and FX, optionally emicizumab, even where the patient has not (yet) developed inhibitory antibodies. Similarly, emicizumab or other bispecific antigen binding molecules for FIXa and FX, and other FVIIIa-activity replacing polypeptide drugs generally, may be administered to patients who were previously treated with a bispecific antigen binding molecule of the present invention. Regiments of treatment may comprise administration of a first FVIII-activity replacing polypeptide drug for a first period (e.g., between one and six months, or between six months and one year), followed by switching to a different FVIII-activity replacing polypeptide drug for a second period (e.g. between one and six months, or between six months and one year), followed by switching back to the first drug or switching to yet another FVIII-activity replacing polypeptide drug. The different amino acid sequences of the different drug treatments should ensure that a patient at risk of developing inhibitory antibodies to one drug is no longer at risk of developing inhibitory antibodies to the first drug (e.g., emicizumab) following switching to a different drug (e.g., a molecule of the present invention). The cycling period may be varied or shortened, according to convenience and the preferences of the patient and doctor.

It will be recognised that administration of the encoding nucleic acid represents an alternative therapy, and may be performed in place of administering the polypeptide drug directly.

As noted, the bispecific antigen-binding molecules of the present invention are believed to have a strong safety profile, associated with no (or minimal) incidents of hypersensitivity reactions, development of allo-antibodies, organ toxicity or other adverse events leading to discontinuation of the therapy.

Clauses

The following numbered clauses represent embodiments of the invention and are part of the description.

1. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein

the first VH domain comprises a set of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408, and/or wherein the first VH domain is at least 95% identical to the N1280H VH domain at the amino acid sequence level;

the second VH domain is at least 95% identical to the T0201H VH domain SEQ ID NO: 470 at the amino acid sequence level, and

the first VL domain and the second VL domain each comprise a set of LCDRs comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences defined wherein LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7 and LCDR3 is SEQ ID NO: 8, and/or wherein the first VL domain and the second VL domain are at least 95% identical to the 0128L VL domain SEQ ID NO: 10 at the amino acid sequence level.

2. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein

the first VH domain is a product of recombination of human immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is IGHV3-7 (e.g., VH3-7*01) and the j gene segment is IGHJ6 (e.g., JH6*02),

the second VH domain is a product of recombination of human immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is IGHV1-46 (e.g., VH1-46*03) and the j gene segment is IGHJ1 (e.g., JH1*01), and optionally wherein the d gene segment is IGHD6-6 (e.g., DH6-6*01), and

the first VL domain and the second VL domain are both products of recombination of human immunoglobulin light chain v and j gene segments, wherein the v gene segment is IGLV3-21 (e.g., VL3-21*d01) and the j gene segment is IGLJ2 (e.g., JL2*01) or IGLJ3 (e.g., JL3*02).

3. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein

the first VH domain has at least 95% amino acid sequence identity with the N1280H VH domain SEQ ID NO: 443,

the second VH domain has at least 95% amino acid sequence identity with the T0201H VH domain SEQ ID NO: 470, and

the first VL domain and the second VL domain each have at least 95% amino acid sequence identity with the 0128L VL domain SEQ ID NO: 10.

4. Bispecific antibody according to any preceding clause, wherein the first VH domain comprises a set of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408. 5. Bispecific antibody according to clause 4, wherein the first VH domain comprises HCDR1 SEQ ID NO: 441. 6. Bispecific antibody according to clause 4 or clause 5, wherein the first VH domain comprises HCDR2 SEQ ID NO: 634. 7. Bispecific antibody according to any of clauses 4 to 6, wherein the first VH domain comprises HCDR2 SEQ ID NO: 436. 8. Bispecific antibody according to any of clauses 4 to 7, wherein the first VH domain comprises HCDR3 SEQ ID NO: 635. 9. Bispecific antibody according to any of clauses 4 to 8, wherein the first VH domain comprises HCDR3 SEQ ID NO: 433. 10. Bispecific antibody according to any preceding clause, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1280H. 11. Bispecific antibody according to any preceding clause, wherein the the first VH domain comprises a set of N1280H HCDRs comprising N1280H HCDR1 SEQ ID NO: 441, N1280H HCDR2 SEQ ID NO: 436 and N1280H HCDR3 SEQ ID NO: 433. 12. Bispecific antibody according to clause 10 or clause 11, wherein the first VH domain is the N1280H VH domain SEQ ID NO: 443. 13. Bispecific antibody according to any of clauses 1 to 11, wherein the first VH domain is the N1454H VH domain SEQ ID NO: 454. 14. Bispecific antibody according to any of clauses 1 to 11, wherein the first VH domain is the N1441H VH domain SEQ ID NO: 456. 15. Bispecific antibody according to any of clauses 1 to 11, wherein the first VH domain is the N1442H VH domain SEQ ID NO: 458. 16. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1333H. 17. Bispecific antibody according to clause 16, wherein the first VH domain comprises a set of N1333H CDRs comprising N1333H CDR1, N1333H CDR2 and N1333H CDR3. 18. Bispecific antibody according to clause 16 or clause 17, wherein the first VH domain is the N1333H VH domain. 19. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1327H. 20. Bispecific antibody according to clause 19, wherein the the first VH domain comprises a set of N1327H HCDRs comprising N1327H HCDR1, N1327H HCDR2 and N1327H HCDR3. 21. Bispecific antibody according to clause 19 or clause 20, wherein the first VH domain is the N1327H VH domain. 22. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1314H. 23. Bispecific antibody according to clause 22, wherein the the first VH domain comprises a set of N1314H HCDRs comprising N1314H HCDR1, N1314H HCDR2 and N1314H HCDR3. 24. Bispecific antibody according to clause 22 or clause 23, wherein the first VH domain is the N1314H VH domain. 25. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1172H. 26. Bispecific antibody according to clause 25, wherein the the first VH domain comprises a set of N1172H HCDRs comprising N1172H HCDR1, N1172H HCDR2 and N1172H HCDR3. 27. Bispecific antibody according to clause 25 or clause 26, wherein the first VH domain is the N1172H VH domain. 28. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1091H. 29. Bispecific antibody according to clause 28, wherein the first VH domain comprises a set of N1091H HCDRs comprising N1091H HCDR1, N1091H HCDR2 and N1091H HCDR3. 30. Bispecific antibody according to clause 28 or clause 29, wherein the first VH domain is the N1091H VH domain. 31. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N0511H. 32. Bispecific antibody according to clause 31, wherein the first VH domain comprises a set of N0511H HCDRs comprising N0511H HCDR1, N0511H HCDR2 and N0511H HCDR3. 33. Bispecific antibody according to clause 31 or clause 32, wherein the first VH domain is the N0511H VH domain. 34. Bispecific antibody according to any of clauses 1 to 3, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N0436H. 35. Bispecific antibody according to clause 34, wherein the the first VH domain comprises a set of N0436H HCDRs comprising N0436H HCDR1, N0436H HCDR2 and N0436H HCDR3. 36. Bispecific antibody according to clause 34 or clause 35, wherein the first VH domain is the N0436H VH domain. 37. Bispecific antibody according to any preceding clause, wherein the second VH domain has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to T0201H VH domain SEQ ID NO: 470. 38. Bispecific antibody according to clause 37, wherein the second VH domain comprises an HCDR1 which is the T0201H HCDR1 SEQ ID NO: 462, an HCDR2 which is the T0201H HCDR2 SEQ ID NO: 467, and/or an HCDR3 which is the T0201H HCDR3 SEQ ID NO: 468. 39. Bispecific antibody according to any preceding clause wherein the second VH domain comprises HCDR1 SEQ ID NO: 636. 40. Bispecific antibody according to clause 39, wherein the second VH domain comprises HCDR1 SEQ ID NO: 598. 41. Bispecific antibody according to any preceding clause, wherein the second VH domain comprises HCDR2 SEQ ID NO: 467. 42. Bispecific antibody according to any preceding clause, wherein the second VH domain comprises HCDR3 SEQ ID NO: 637. 43. Bispecific antibody according to clause 42, wherein the second VH domain comprises HCDR3 SEQ ID NO: 638. 44. Bispecific antibody according to clause 43, wherein the second VH domain comprises HCDR3 SEQ ID NO: 639. 45. Bispecific antibody according to clause 44, wherein the second VH domain comprises HCDR3 SEQ ID NO: 565. 46. Bispecific antibody according to clause 44, wherein the second VH domain comprises HCDR3 SEQ ID NO: 583. 47. Bispecific antibody according to any of clauses 37 to 45, wherein the second VH domain comprises SEQ ID NO: 632. 48. Bispecific antibody according to any of clauses 37 to 45, wherein the second VH domain comprises SEQ ID NO: 600. 49. Bispecific antibody according to any of clauses 37 to 46, wherein the second VH domain comprises SEQ ID NO: 585. 50. Bispecific antibody according to clause 38, wherein the the second VH domain comprises a set of T0201H HCDRs comprising T0201H HCDR1, T0201H HCDR2 and T0201H HCDR3. 51. Bispecific antibody according to clause 37, clause 38 or clause 50, wherein the second VH domain is the T0201H VH domain, optionally with a substitution at Cys114. 52. Bispecific antibody according to clause 51, wherein the substitution at Cys114 is Ile, Gln, Arg, Val or Trp. 53. Bispecific antibody according to any of clauses 1 to 38, wherein the the second VH domain comprises a set of T0638H HCDRs comprising T0638H HCDR1, T0638H HCDR2 and T0638H HCDR3. 54. Bispecific antibody according to clause 53, wherein the second VH domain is the T0638 VH domain, optionally with a substitution at Cys114. 55. Bispecific antibody according to clause 54, wherein the substitution at Cys114 is Ile, Gln,

Arg, Val or Trp.

56. Bispecific antibody according to any preceding clause, wherein the first VL domain and the second VL domain each have at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with 0128L SEQ ID NO: 10. 57. Bispecific antibody according to any preceding clause, wherein the first VL domain and the second VL domain each comprise a set of 0128L CDRs comprising 0128L LCDR1 SEQ ID NO: 6, 0128L LCDR2 SEQ ID NO: 7 and 0128L LCDR3 SEQ ID NO: 8. 58. Bispecific antibody according to any preceding clause, wherein the first VL domain and the second VL domain are identical in amino acid sequence. 59. Bispecific antibody according to clause 58, wherein the first VL domain and the second VL domain comprise the 0325L amino acid sequence SEQ ID NO: 416. 60. Bispecific antibody according to clause 58 or clause 59, wherein the first VL domain and the second VL domain comprise the 0128L amino acid sequence SEQ ID NO: 10. 61. Bispecific antibody according to any preceding clause, wherein each heavy-light chain pair further comprises a CL constant domain paired with a CH1 domain. 62. Bispecific antibody according to any preceding clause, wherein the heavy-light chain pairs comprise a common light chain. 63. Bispecific antibody according to clause 62, wherein the common light chain comprises the CL amino acid sequence SEQ ID NO: 146 of the 0128L light chain. 64. Bispecific antibody according to clause 63, wherein the common light chain is the 0325L light chain SEQ ID NO: 414. 65. Bispecific antibody according to clause 63, wherein the common light chain is the 0128L light chain SEQ ID NO: 405. 66. Bispecific antibody according to any preceding clause, wherein the heavy chain of each heavy-light chain comprises a heavy chain constant region and wherein the first and second heavy-light chain pairs associate to form tetrameric immunoglobulin through dimerisation of the heavy chain constant regions. 67. Bispecific antibody according to clause 66, wherein the heavy chain constant region of the first heavy-light chain pair comprises a different amino acid sequence from the heavy chain constant region of the second heavy-light chain pair, wherein the different amino acid sequences are engineered to promote heterodimerisation of the heavy chain constant regions. 68. Bispecific antibody according to clause 67, wherein the heavy chain constant regions comprise knobs-into-holes mutations or charge pair mutations. 69. Bispecific antibody according to clause 67, wherein the heavy chain constant region of one (e.g., the first) heavy-light chain pair is a human IgG4 constant region comprising substitution K439E and wherein the heavy chain constant region of the other (e.g., the second) heavy-light chain pair is an IgG4 region comprising substitution E356K, wherein constant region numbering is according to the EU numbering system. 70. Bispecific antibody according to any of clauses 66 to 69, wherein the heavy chain constant region of one or both heavy-light chain pairs is a human IgG4 constant region comprising substitution S228P, wherein constant region numbering is according to the EU numbering system. 71. Bispecific antibody according to any of clauses 66 to 70, wherein the heavy chain constant region of one (e.g., the first) heavy-light chain pair comprises SEQ ID NO: 409 and the heavy chain constant region of the other (e.g., the second) heavy-light chain pair comprises SEQ ID NO: 410. 72. Bispecific antibody according to any of clauses 66 to 71, comprising

a first heavy chain comprising a first VH domain amino acid sequence SEQ ID NO: 443 or SEQ ID NO: 456,

a second heavy chain comprising a second VH domain amino acid sequence SEQ ID NO: 632, and

a common light chain comprising a VL domain amino acid sequence SEQ ID NO: 416.

73. Bispecific antibody according to any of clauses 66 to 72, comprising

a first heavy chain comprising amino acid sequence SEQ ID NO: 419,

a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and

a common light chain comprising amino acid sequence SEQ ID NO: 414.

74. Bispecific antibody according to any of clauses 66 to 71, comprising

a first heavy chain comprising amino acid sequence SEQ ID NO: 424

a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and

a common light chain comprising amino acid sequence SEQ ID NO: 414.

75. Bispecific antibody according to any of clauses 66 to 72, comprising

a first heavy chain comprising amino acid sequence SEQ ID NO: 426

a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and

a common light chain comprising amino acid sequence SEQ ID NO: 414.

76. Bispecific antibody according to any of clauses 66 to 71, comprising

a first heavy chain comprising amino acid sequence SEQ ID NO: 428

a second heavy chain comprising amino acid sequence SEQ ID NO: 430, and

a common light chain comprising amino acid sequence SEQ ID NO: 414.

77. Bispecific antibody according to any of clauses 66 to 71, comprising

a first heavy chain comprising amino acid sequence SEQ ID NO: 428

a second heavy chain comprising amino acid sequence SEQ ID NO: 432, and

a common light chain comprising amino acid sequence SEQ ID NO: 414.

78. Bispecific antibody according to clause 66, wherein the heavy chain constant region of the first heavy-light chain pair is identical to the heavy chain constant region of the second heavy-light chain pair. 79. Bispecific antibody according to any of clauses 1 to 68, wherein the antibody is human IgG. 80. Bispecific antibody according to clause 79, wherein the antibody is human IgG4. 81. Bispecific antibody according to clause 79 or clause 80, wherein the IgG comprises the IgG4-PE heavy chain constant region SEQ ID NO: 143, optionally engineered with one or more amino acid substitutions to promote heterodimerisation. 82. Bispecific antibody according to clause 79 or clause 80, wherein the antibody comprises the Fc region of emicizumab. 83. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1280H VH domain SEQ ID NO: 443, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the T0999H VH domain SEQ ID NO: 632, and wherein

the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO: 414.

84. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1454H VH domain SEQ ID NO: 454, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the T0999H VH domain SEQ ID NO: 632, and wherein

the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO: 414.

85. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1441H VH domain SEQ ID NO: 456, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the T0999H VH domain SEQ ID NO: 632, and wherein

the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO: 414.

86. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1442H VH domain SEQ ID NO: 458, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the T0736H VH domain SEQ ID NO: 600, and wherein

the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO: 414.

87. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1442H VH domain SEQ ID NO: 458, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the T0687H VH domain SEQ ID NO: 585, and wherein

the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO: 414.

88. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein

a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1333H VH domain, and

a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the T0638H VH domain, and wherein

the first and second heavy-light chain pairs each comprise a common light chain comprising the 0128L light chain amino acid sequence SEQ ID NO: 405.

89. Bispecific antibody according to any preceding clause, which reduces the coagulation time of FVIII-deficient human blood plasma to less than 40 seconds in an aPTT assay. 90. Bispecific antibody according to clause 89, which reduces the coagulation time of FVIII-deficient human blood plasma to less than 30 seconds in an aPTT assay. 91. Bispecific antibody according to clause 90, which reduces the coagulation time of FVIII-deficient human blood plasma to 22-28 seconds in an aPTT assay. 92. Bispecific antibody according to clause 91, which reduces the coagulation time of FVIII-deficient human blood plasma to 24-26 seconds in an aPTT assay. 93. Bispecific antibody according to any preceding clause, which enhances the FIXa-mediated activation of FX to FXa to the same or similar extent as emicizumab in a FXase assay. 94. Bispecific antibody according to any preceding clause, which enhances the FIXa-mediated activation of FX to FXa to at least the same extent as emicizumab. 95. Bispecific antibody according to any of clauses 89 to 94, wherein said coagulation time or FIXa-mediated activation is as determined at an antibody concentration of 0.1 mg/ml, 0.3 mg/ml or 0.5 mg/ml at 37 degrees C. 96. Bispecific antibody according to any preceding clause, wherein the antibody has an EC50 for Cmax in a fluorometric thrombin generation assay (TGA) that is within 10% of or is lower than the Cmax EC50 of emicizumab in said assay, and/or wherein the antibody generates a maximal response of Cmax between 200 and 450 nM thrombin in a fluorometric TGA. 98. Bispecific antibody according to clause 96, wherein the antibody has an EC50 of less than 50 nM for Cmax in a fluorometric TGA. 99. Bispecific antibody according to clause 98, which has an EC50 of less than 10 nM for Cmax in a fluorimetric TGA. 100. Bispecific antibody according to any preceding clause, wherein the maximal response of Cmax is between 250 nM and 400 nM. 101. Bispecific antibody according to any preceding clause, wherein the antibody has an EC50 for Tmax in a fluorimetric TGA that is within 10% of, or is lower than, the Tmax EC50 of emicizumab in said assay, and/or wherein the antibody generates a maximal response of Tmax between 1 and 10 minutes. 102. Bispecific antibody according to any preceding clause, wherein the antibody has an EC50 of less than 5 nM for Tmax in a fluorimetric TGA. 103. Bispecific antibody according to clause 102, wherein the EC50 for Tmax is less than 2 nM. 104. Bispecific antibody according to any preceding clause, wherein the antibody generates a maximal response of Tmax between 2 and 8 minutes in a fluorimetric TGA. 105. Anti-FIXa antibody comprising two copies of the first heavy-light chain pair as defined in any preceding clause. 106. Anti-FX antibody comprising two copies of the second heavy-light chain pair as defined in any of clauses 1 to 104. 107. Isolated nucleic acid encoding an antibody according to any preceding clause. 108. A host cell in vitro comprising recombinant DNA encoding

an antibody heavy chain comprising a first VH domain as defined in any of clauses 1 to 104,

an antibody heavy chain comprising a second VH domain as defined in any of clauses 1 to 104, and/or

an antibody light chain comprising a first or second VL domain as defined in any of clauses 1 to 104.

109. A host cell according to clause 108 comprising recombinant DNA encoding

a first heavy chain comprising amino acid sequence SEQ ID NO: 419 or SEQ ID NO: 426,

a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and

a common light chain comprising amino acid sequence SEQ ID NO: 414.

110. A population of host cells in vitro, wherein each host cell comprises recombinant DNA encoding a bispecific antibody according to any of clauses 1 to 104. 111. A kit for production of a bispecific antibody according to any of clauses 1 to 104, comprising

an antibody heavy chain comprising a first VH domain as defined in any of clauses 1 to 104, or nucleic acid encoding said heavy chain,

an antibody heavy chain comprising a second VH domain as defined in any of clauses 1 to 104, or nucleic acid encoding said heavy chain,

an antibody light chain comprising a first VL domain as defined in any of clauses 1 to 104, or nucleic acid encoding said light chain, and

an antibody light chain comprising a second VL domain as defined in any of clauses 1 to 104, or nucleic acid encoding said light chain.

112. A kit according to clause 111, comprising

a first heavy chain comprising amino acid sequence SEQ ID NO: 419 or SEQ ID NO: 426, or nucleic acid encoding said first heavy chain,

a second heavy chain comprising amino acid sequence SEQ ID NO: 421, or nucleic acid encoding said second heavy chain, and

a common light chain comprising amino acid sequence SEQ ID NO: 414, or nucleic acid encoding said common light chain.

113. A kit according to clause 111 or clause 112, wherein said amino acid sequences or said nucleic acids are provided in cells. 114. A kit according to clause 111 or clause 112, wherein said amino acid sequences or said nucleic acids are provided in cell-free buffered aqueous media. 115. A kit according to any of clauses 111 to 114, wherein each of said amino acid sequences or each of said nucleic acids is provided in a separate phial. 116. A method of producing a bispecific antibody according to any of clauses 1 to 104, comprising culturing host cells according to clause 108 or clause 109 under conditions for expression of the bispecific antibody, and recovering the bispecific antibody from the host cell culture. 117. A method according to clause 116, comprising culturing the host cells in a vessel comprising a volume of at least 100 litres. 118. A method according to clause 117, wherein the vessel is of stainless steel or is a single-use bioreactor. 119. A composition comprising a bispecific antibody according to any of clauses 1 to 104, or isolated nucleic acid according to clause 107, in solution with a pharmaceutically acceptable excipient. 120. A composition according to clause 119, wherein the bispecific antibody or nucleic acid is in sterile aqueous solution. 121. A composition according to clause 119 or clause 120, comprising a bispecific antibody according to any of clauses 1 to 104 wherein the bispecific antibody is at least 95% pure such that the composition comprises no more than 5% homodimeric antibody contaminants. 122. A composition according to clause 121, wherein the bispecific antibody is at least 99% pure such that the composition comprises no more than 1% homodimeric antibody contaminants. 123. A method of controlling bleeding in a patient, comprising administering a composition according to any of clauses 119 to 122 to the patient. 124. A composition according to any of clauses 119 to 122 for use in a method of treatment of the human body by therapy. 125. A composition according to any of clauses 119 to 122 for use in a method of controlling bleeding in a patient. 125. Use of a bispecific antibody according to any of clauses 1 to 104 for the manufacture of a medicament for controlling bleeding in a haemophilia A patient. 126. A method according to clause 123, or a composition for use or use according to clause 125, wherein the patient is a haemophilia A patient. 127. A method or a composition for use according to clause 126, wherein the patient is resistant to treatment with FVIII owing to the presence of inhibitory antibodies in the bloodstream. 128. A method or a composition for use according to clause 126 or clause 127, wherein the patient is resistant to treatment with another bispecific antibody to FIXa and FX owing to the presence of inhibitory antibodies in the bloodstream. 129. A method or a composition for use according to clause 128, wherein the patient is resistant to treatment with emicizumab. 130. A method of reducing development of inhibitory anti-drug antibodies in a haemophilia A patient undergoing treatment with a polypeptide that replaces FVIlla activity, comprising

administering a first FVIIIa-activity replacing polypeptide drug to the patient for a period of 1-12 months,

switching the patient to a second, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, and

switching the patient to either the first antigen-binding molecule or to a third, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, wherein

the first, second or third FVIIIa-activity replacing polypeptide drug is a bispecific antibody according to any of clauses 1 to 104,

and wherein in each case the FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid is administered in a therapeutically effective amount to functionally replace FVIIIa in the patient, and wherein the risk of the patient developing inhibitory anti-drug antibodies to any of the FVIIIa-activity replacing polypeptide drug is reduced compared with a patient continuing to receive treatment with that FVIIIa-activity replacing polypeptide drug.

131. A composition comprising a FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid, for use in a method of treating a haemophilia A patient while reducing development of inhibitory anti-drug antibodies, or use of a FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid for the manufacture of a medicament for use in a method of treating a haemophilia A patient while reducing development of inhibitory anti-drug antibodies, the method comprising

administering a first FVIIIa-activity replacing polypeptide drug to the patient for a period of 1-12 months,

switching the patient to a second, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, and

switching the patient to either the first antigen-binding molecule or to a third, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, wherein

the first, second or third FVIIIa-activity replacing polypeptide drug is a bispecific antibody according to any of clauses 1 to 104,

and wherein in each case the FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid is administered in a therapeutically effective amount to functionally replace FVIIIa in the patient, and wherein the risk of the patient developing inhibitory anti-drug antibodies to any of the FVIIIa-activity replacing polypeptide drug is reduced compared with a patient continuing to receive treatment with that FVIIIa-activity replacing polypeptide drug.

132. A method according to clause 130, or a composition for use or use according to clause 131, wherein the first, second and third FVIIIa-activity replacing polypeptide drugs are recombinant or plasma-derived FVIII, emicizumab, and a bispecific antibody according to any of clauses 1 to 104, in any order. 133. A method, composition for use or use according to any of clauses 123 to 132 wherein the treatment comprises subcutaneous administration of the composition to the patient.

Equivalents: Those skilled in the art will recognise, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be within the scope of protection of the appended claims.

EXAMPLES

Bispecific IgG antibodies comprising Fv binding sites for human FIXa and human FX were generated as described in PCT/EP2018/066836 filed on 22 Jun. 2018 entitled “Bispecific antibodies for factor IX and factor X” (WO2018/234575). As described therein, an extensive campaign of immunisation and screening led to the identification of an anti-FIXa antibody NINA-0128 which, when paired in IgG format with any of a selection of different anti-FX binding Fvs, showed outstanding activity in functional screens including a tenase assay and aPTT assay. NINA-0128 comprises VH domain N0128H and VL domain 0128L. A number of variants of the N0128H VH domain were generated and tested, resulting in further improvements in function in a bispecific format, including for example the N0436H VH domain.

Building on that work, bispecific IgG were designed with the VL domain of NINA-0128 as a common light chain. A panel of anti-FX antibodies were generated in vivo in a transgenic mouse comprising human immunoglobulin genes. These were co-expressed with NINA-0128 as the anti-FIX binding arm, using the 0128L VL domain in a common light chain including a human constant region. One VH domain, T0200, showed outstanding activity in the bispecific format and was selected for further development. Structurally related antibodies obtained from the same immunised animal as the T0200H clone, included further anti-FX VH domains that performed even better than the T0200H VH domain in bispecific IgG4 with an anti-FIX VH domain and the 0128L common light chain.

Meanwhile, further anti-FIXa antibody variants were generated, introducing mutations in the VH domain while retaining the common 0128L VL domain. The anti-FIXa N0436H VH domain sequence was optimised by substituting all possible amino acids at each position in CDR1, CDR2 and CDR3, expressing the resulting VH domain variants in the context of bispecific antibodies comprising the common light chain, evaluating the variant bispecific antibodies in a range of functional assays, identifying mutations associated with increased functional activity, and generating further variants including combinations of mutations associated with increased functional activity.

Improved T0200H VH domain variants were combined with improved N0436H VH domain variants, each paired with the N0128L common light chain, and repeated rounds of optimisation, screening and selection were conducted.

FIG. 6 shows a simplified overview of the screening program.

Very strong FVIII mimetic activity was achieved with common light chain bispecific antibodies including the optimised sequences. The following bispecific antibodies are examples of strong performers, as indicated by functional characterisation in a range of disease-relevant assays. Nomenclature of the bispecific antibodies which have a common light chain is IXAX-nnnn.tttt.llll, wherein nnnn is a 4 digit numerical identifier of the anti-FIX VH domain, tttt is a 4 digit identifier of the anti-FX VH domain, and 1111 is a 4 digit numerical identifier of the common VL domain:

IXAX-0128.0201.0128 (anti-FIXa VH domain N0128H; anti-FX VH domain T0201H; 0128L common light chain)

IXAX-0436.0201.0128

IXAX-0511.0201.0128

IXAX-1091.0201.0128

IXAX-1172.0201.0128

IXAX-1280.0201.0128

IXAX-1341.0201.0128

IXAX-1327.0201.0128

IXAX-1333.0201.0128

Other high performing anti-FX VH domains which combine well with the above and other anti-FIX VH domains in bispecific antibodies were those of the T0201H lineage and variants thereof such as those listed in FIG. 11 and other related sequences. Particularly good results were obtained with bispecific antibodies including an anti-FIX VH domain, an anti-FX VH domain and a common light chain selected from the following:

anti-FIX VH domain: anti-FX VH domain: common VL domain: N0436H T0201H 0128L N0511H T0596H 0325L N1091H T0616H N1172H T0638H N1280H T0666H N1327H T0678H N1333H T0681H N1341H T0687H N1441H T0736H N1442H T0999H N1454H

For example,

IXAX-1280.0999.0325

IXAX-1454.0999.0325

IXAX-1441.0999.0325

IXAX-1441.0736.0325

IXAX-1442.0687.0325

The bispecific antibodies described here represent candidate pharmaceutical drug molecules for therapeutic use as described herein. They may offer a vital healthcare option for patients by providing an alternative to existing treatments such as emicizumab, especially in patients for whom such existing treatments are no longer effective due to the presence of anti-drug antibodies.

In these Examples, the reference antibody AbE or Antibody E is a bispecific antibody having the heavy and light chain amino acid sequences of emicizumab [3].

Example 1. Creation of Anti-FX Antibody Panel with Common Light Chain

Transgenic mice expressing a common light chain comprising the 0128L VL domain of were immunised with human factor X. Antigen specific B cells were single cell sorted by flow cytometry and the VH and VL sequences were retrieved by next generation sequencing (NGS). 200 anti-FX heavy chains were identified by NGS analysis of the single cell sorted lymphocytes. Further bulk NGS analysis was performed on bone marrow and lymph node tissues harvested from the same transgenic animals.

Example 2. Creation of Anti-FIXaxFIX Bispecific Antibodies with Common Light Chain

Each anti-FX heavy chain was expressed in HEK293 cells as bispecific antibody comprising the anti-FIX N0128H heavy chain and the 0128L common light chain. The bispecific antibodies were purified by Protein A substantially as described in PCT/EP2018/066836 filed on 22 Jun. 2018 entitled “Bispecific antibodies for factor IX and factor X”, which is incorporated by reference herein.

Example 3. Initial Screening of Anti-FX Arms Using Activation Coagulation Factor VIII (FVIIIa)-Like Activity Assay (FXase or Tenase Assay)

200 bispecific antibodies comprising a range of different anti-FX heavy chains, each in combination with the N0128H anti-FIX heavy chain and 0128L common VL domain, were screened using a factor Xa generation assay. This functional screening detects FVIIIa-mimetic activity, i.e., ability to enhance (catalyse) the FIXa-mediated activation of FX to FXa, in vitro by enzymatic “FXase” assay. In this assay, the test bispecific molecule is contacted with FIXa and FX in the presence of phospholipid, under conditions suitable for formation of FXa. A substrate for FXa is added which, when cleaved by FXa, generates a detectable product. Detection of this product in the presence of test bispecific antibody is compared with a negative control in which no test antibody is present (a control antibody may be included). The detected signal is quantified by recording absorbance of the reaction solution at 405 nm. Absorbance is measured across a range of antibody concentrations in the assay and an EC50 value is calculated as a measure of the bispecific antibody potency in this assay. Significant difference of EC50 between test antibody and control indicates that the test antibody is able to enhance FIXa-mediated activation of FX. FIG. 7.

Results

Among all the bispecific antibodies assayed, a single one showed outstanding FXase activity: the N0128H anti-FIX heavy chain and T0200H anti-FX heavy chain, paired with 0128L common VL domain, had markedly higher FXase activity than all others in the panel. FIG. 8.

Materials & Methods—Standard FXase Reaction Conditions

7.5 μL FIX (3.75 μg/mL) and 5 μL supernatant from the Expi293 cells producing the recombinant antibodies (Example 8) were added to each well of an assay plate and incubated at room temperature for 1 hour. A mixture of 2.5 μL FXIa (10 ng/mL), 5 μL FX (50 ng/mL), 0.05 μL phospholipid (10 mg/mL) and 5 μL TBSB-S buffer was added to each well to initiate enzymatic reaction (FIXa cleavage of FX to generate FXa), and incubated at 37° C. for 1 hour. After 60 minutes, the reaction was terminated by adding 5 μL of 0.5 M EDTA. After adding 10 μL S2765 substrate solution to each well, absorbance at 405 nm (reference wavelength 655 nm) was measured for 30 minutes (one reading per 10 minutes). All reactions were performed at 37° C. unless otherwise stated.

TBSB:

Tris buffered saline containing 0.1% bovine serum albumin To make 7.5 mL TBSB: 0.1 mL 7.5% BSA solution (Sigma) 7.4 mL 1×TBS solution (diluted from 20×TBS solution ThermoFisher)

TBSB-S:

TBSB containing 5 mM CaCl2) and 1 mM MgCl2 To make 100 mL TBSB-S:

99.4 mL TBSB 0.5 mL 1M CaCl₂) (Sigma) 0.1 mL 1M MgCl2 (Sigma)

FXIa Stock Solution (10 μg/mL): Add 10 mL TBSB-S to 0.1 mg FXIa (Enzyme Research Laboratories) to make 10 μg/mL stock solution. Dilute to 10 ng/mL (1:1,000) working solution before use. F.IXa Stock Solution (5 μg/mL) Add 100 mL TBSB-S to 0.5 mg FIXa (HFIXa 1080) (Enzyme Research Laboratories) to make 5 μg/mL stock solution. Dilute to 1.0 μg/mL (1:5) working solution before use. FIX Stock Solution (37.5 μg/mL): Add 13.3 mL TBSB-S to 0.5 mg FIX (Enzyme Research Laboratories) to make 37.5 μg/mL stock solution. Dilute to 3.75 μg/mL (1:10) working solution before use. FX Working Solution (50 μg/mL): Add 16 mL TBSB-S to 0.8 mg FX (Enzyme Research Laboratories) to make 50 μg/mL working solution. No further dilution is needed before use.

S2765 Stock Solution:

25 mg S2765 (Chromogenix) chromogenic substrate (0.035 mmol) To make 2 mM stock solution: Add 17.493 mL water to the vial and dissolve with shaking.

Polybrene Solution:

To make 0.6 g/L hexadimethrine bromide stock solution: Add 0.15 g hexadimethrine bromide (Sigma) to 250 mL water. Dilute to 0.6 mg/L (1:1,000) working solution before use.

S2765 Substrate Working Solution

A 1:1 mixture of 2 mM S-2765 stock solution and 0.6 mg/L polybrene solution.

Example 4. Identification of Anti-FX T0200H VH Domain

The bispecific antibody designated IXAX.0128.0200.0128, comprising N0128H anti-FIX VH domain, T0200H anti-FX VH domain and 0128L common light chain, demonstrated high FXase activity compared with the other bispecific antibodies. The T0200H VH domain was chosen for further development to attempt production of yet further improved bispecific antibodies.

Example 5. Optimisation of Anti-FX T0200H VH Domain Phylogenetic Analysis

From the bulk NGS (Example 1) and phylogenetic analysis, 113 anti-FX heavy chains were identified as belonging to the same lymphocyte cluster as the anti-FX heavy chain T0200H. The cluster represents B cells that appear to share a common evolutionary lineage. The anti-FX heavy chains within the cluster shared approximately 95% sequence identity with T0200H at the amino acid level. FIG. 9.

The 113 anti-FX heavy chains were expressed in bispecific antibodies with a panel of different anti-FIX heavy chains and the 0128L common light chain, and screened by FXase assay.

We identified several anti-FX-heavy chains that showed increased FXase activity compared with the T0200H VH domain when assayed as bispecific antibodies. FIG. 10 shows example data from the Xase assay.

A selection of the most active FX arm sequences is shown in FIG. 11. These VH domains were designated T0201H to T0217H respectively. Those with the strongest activity in bispecific format were T0204, T0207, T0205 and T0201 (FIG. 10b ).

Using amino acid sequence comparisons, and supported by the functional data, we identified several amino acid residues in frameworks and CDR regions of the anti-FX heavy chains that differ in the most active VH domains and may contribute to the enhanced biological activity compared with T0200H. For example, one or more of the following amino acid features of the VH domain may increase the FVIII-mimetic activity of bispecific antibodies containing the VH domain (IMGT residue numbering):

Replacement of valine (V) by isoleucine (I) at position 5 in FR1;

Replacement of lysine (K) by glutamine (Q) at position 13 in FR1;

Replacement of leucine (L) by methionine (M) at position 39 in FR2;

Replacement of threonine (T) by serine (S) at position 62 in CDR2;

Replacement of aspartate (D) by serine (S) at position 64 in CDR2;

Replacement of threonine (T) by serine (S) at position 85 in FR3;

Replacement of alanine (A) by serine (S) at position 112 in the CDR3.

Nevertheless it is clear that activity is high even without these amino acid substitutions, since T0200H itself shows strong activity in a bispecific antibody, and none of these substitutions was consistently present in all of the top VH domains (FIG. 11).

Targeted Mutagenesis for Functional Optimisation

The CDR3 of VH domain T0201H was systematically mutated to provide a library of VH domains in which the residue at each position was individually replaced by another amino acid. The resulting VH domains were named T0XXXH, where XXX numbers are shown in FIG. 12 for the mutants of IMGT positions 114 (Cys), 115 (Leu), 116 (Gin) and 117 (Leu). Refer to FIG. 13 for IMGT numbering.

Removal of Potential Developmental Liability

An unpaired cysteine (C) residue present in CDR3 was identified as a high-risk sequence motif. This unpaired cysteine, present at position 114 in the CDR3 of T0200H and all 113 further anti-FX VH domains identified from the bulk NGS analysis, represents a liability for the development of the bispecific antibody. We screened VH domains containing substitutions of all other amino acids for the cysteine at this position in T0201H. These new variants were expressed with N1280H (see Example 6) and 0128L common light chain as IgG4 bispecific antibodies, purified by Protein A and screened for FVIII mimetic activity by FXase assay. Replacement of cysteine at position 114 with isoleucine (I), glutamine (Q), arginine (R), valine (V) or tryptophan (W) resulted in bispecifics antibodies with FVIII mimetic activity similar to bispecific antibodies having the T0201H or T0202H VH domains. We conclude C114 can be replaced with a variety of other amino acids and still maintain FVIII mimetic activity.

Example 6. Systematic Sequence Optimisation of Anti-FIX VH

Each amino acid residue in CDR1, CDR2 and CDR3 was individually mutated to generate single position mutants of the anti-FIX N0128H heavy chain. The anti-FIX heavy chain variants thus generated were expressed in bispecific format, paired with anti-FX heavy chain T0201H and N0128L common VL domain in HEK293.

Protein A purified bispecific antibodies were assayed for biological activity by FXase (Example 7) and aPTT to look for amino acid changes that improved the FVIII-mimetic activity of the bispecific antibody. Improved variants were then combined to generate double or triple mutants in the CDR1, CDR2 and CDR3 regions.

Table N identifies mutants of the N0128H VH domain in which one or more residues of the CDRs are mutated to other amino acids. For example the N0436H VH domain is a Ser->Ile mutant of the N0128H VH domain, i.e., in which the serine at IMGT position 111A in CDR3 is replaced by isoleucine. Further residue mutations were introduced on top of initial single mutations. For example the N0511H VH domain is a Ser112ALys mutant of the N0436H VH domain, i.e., in which the serine at IMGT position 112A in CDR3 is replaced by lysine. N1172H is a Glu64Arg mutant of the N0511H VH domain, i.e., in which the glutamate at IMGT position 64 in CDR2 is replaced by arginine. N1280H is a Thr29Arg mutant of the N1172H VH domain, i.e., in which the Thr at IMGT position 29 in CDR1 is replaced by arginine. The other named VH domains can be identified from Table N in the same manner.

Refer to FIG. 14 for IMGT numbering.

Example 7. Screening of Improved Bispecific Antibodies in FXase Assay

Anti-FX arms comprising the VH domain variants generated as described in Example 5 were combined with anti-FIX arms comprising the VH domain variants generated as described in Example 6, each paired with the 0128L common VL domain, to generate FIXAxFX bispecific antibodies, and screened for functional activity in the tenase assay.

Results

Example data are shown:

FIG. 10.

FIG. 15.

FIG. 16.

Highly active bispecific antibodies were identified for several combinations of anti-FIX VH and anti-FX VH domains, each paired with the 0128L common VL domain. Examples of anti-FIX VH and anti-FX VH domain combinations are shown in FIG. 16.

The identity of the anti-FX VH domain appeared to have a stronger influence than the identity of the anti-FIX VH domain for these bispecific arm combinations, with T0638H, T0616H, T0596H and T0663H being among the highest-performing anti-FX VH domains. These anti-FX domains performed well in combination with a variety of anti-FIX arms, including variants of N1280H such as those indicated in FIG. 16. Anti-FIX VH domain sequences are identified by reference to appended Table N. Anti-FX VH domain sequences are identified by reference to FIG. 12.

FVIII-mimetic activity of N128 bispecific antibody was sequentially optimised by modifying amino acid residues in any of the three CDRs. Several amino acid residues were identified to increase FVIII mimetic activity across the CDRs and these mutations were combined to maximise activity. The FVIII mimetic activity of antibodies with the N0128H VH domain was progressively improved with further VH domains in the following order: N0128H->N0436H->N0511H->N1091H->N01172H->N1280H->N1333H. FIG. 17.

Materials & Methods—Modified FXase Reaction Conditions.

Initial screening for bispecific antibody FVIII mimetic activity was assessed using the Standard FXase Reaction Conditions set out above in Example 3. As the FVIII mimetic activity of the bispecific antibody increased, the Standard FXase reaction conditions were no longer sufficient to detect improvements in FXase activity. Therefore, more sensitive Modified FXase Reaction Conditions were established.

This modified assay differs from the Standard FXase Reaction Conditions in the following ways: FXIa is not used, the activated form of Factor IX (FIXa) is used and there is no incubation step. All FXase reagents are mixed with a bispecific antibody and the generation of FXa is detected by recording the absorbance of the reaction solution 40 to 50 times every 30 seconds at 405 nm using an Envision plate reader set to 37° C.

18.45 μl TBSB-S buffer was mixed with 0.05 μl phospholipid (10 mg/ml) and mixed vigorously by pipetting to disperse the phospholipid. To this mixture 1.5 μl FIXa (1 μg/ml) and 5 μl of FX (50 μg/ml), was combined with 5 μl of polybrene (0.6 mg/L) and 5 μl S2765 (4 mM), all pre-warmed to 37° C. Finally, 5 μl of bispecific antibody being investigated for FXase activity was added. Absorbance at 405 nm (reference wavelength 655 nm) was recorded 40 to 50 times every 30 seconds.

Example 8. Screening of Improved Bispecific Antibodies in Plasma Coagulation Assay

Anti-FX arms comprising the VH domain variants generated as described in Example 5 were combined with anti-FIX arms comprising the VH domain variants generated as described in Example 6, each paired with the 0128L common VL domain, to generate FIXaxFX bispecific antibodies. To determine the ability of the bispecific antibodies of the present invention to correct the coagulation ability of the blood of haemophilia A patients, the effect of these antibodies on the activated partial thromboplastin time (aPTT) using FVIII deficient plasma was examined.

A mixture of 5 μL of bispecific antibody solution having a variety of concentrations, 20 μL of FVIII deficient plasma (Helena Biosciences), and 25 μL of aPTT reagent (APTT Si L Minus, Helena Biosciences) was warmed at 37° C. for 3 minutes. The coagulation reaction was initiated by adding 25 μL of 25 mM CaCl₂ (Helena Biosciences) to the mixture. The time period until coagulation was measured. Apparatus used for this was C-4 4 channel coagulation analyser (Helena Biosciences).

A sample of results is shown in FIG. 18.

Concentration dependency was subsequently determined for bispecific antibodies that exhibited the highest coagulation time-reducing effect.

For example, IXAX-1280.0201.0128 IgG4 antibody demonstrated a dose dependent decrease in aPTT, comparable to the reference antibody AbE (positive control). FIG. 19. No reduction in aPTT was observed for an isotype control antibody. Note that the antibody preparation used for this assay was the result of a one-step purification on Protein A and as such contained residual anti-FIX monospecific antibodies and residual anti-FX monospecific antibodies in addition to the desired bispecific fraction.

Example 9. Analysis of Anti-FIX VH Domains in Bispecific Antibodies

Considering data from a variety of functional assays including those described in Example 7 and Example 8, it was noted that the anti-FX VH domain T0201 and its sequence variants performed well in combination with a variety of anti-FIX VH domains in bispecific antibodies with the common light chain. For example, anti-FIX VH domains N0128H, N0436H, N0511H, N1091H, N1172H, N1280H, N1314H, N1327H and N1333H all gave good functional activity in the bispecific antibodies. These anti-FIX VH domains share a close structural relationship. FIG. 20. Their performance could be further enhanced by fine tuning of residues through substitution (Table N) and combining substitutions associated with improved activity.

Example 10. Affinity for Antigen-Binding

Binding affinity and the kinetics of antibody-antigen interaction were determined using SPR. Affinity and kinetics of purified test antibodies (all IgG4PE) were compared to comparator anti-FIX antibody AbN or comparator anti-FX antibody AbT as positive control and to an isotype control (ISTC) as negative control.

Binding Affinity for FIX

The anti-FIX antibodies analysed showed binding to FIX in the affinity range of approximately 0.18 μM to 0.3 μM and fast association (k_(on)) and dissociation (k_(off)) rates for FIX. The anti-FIX antibodies analysed showed slightly higher binding affinity to FIX and higher association rate compared to the comparator antibody AbN. No binding to FIX was observed with ISTC. Table E-10-1.

TABLE E-10-1 Binding affinity and kinetic constants on-rate (kon) and off- rate (koff) of anti-FIX antibodies. Anti-FIXa monospecific antibody nomenclature: NINA-hhhh.llll, wherein hhhh is the numeric identifier of the VH domain (e.g., N0436H) and llll is the numeric identifier of the VL domain (e.g., 0128L), Captured anti-FIX antibody k_(on) (1/Ms) k_(off) (1/s) K_(D) (M) NINA-0128 (n = 2 average) 2.92 × 10⁵ 5.76 × 10⁻² 1.98 × 10⁻⁷ NINA-0436.0128 2.46 × 10⁵ 4.53 × 10⁻² 1.84 × 10⁻⁷ NINA-0438.0128 2.30 × 10⁵ 6.73 × 10⁻² 2.93 × 10⁻⁷ NINA-0440.0128 1.85 × 10⁵ 5.35 × 10⁻² 2.89 × 10⁻⁷ NINA-0442.0128 1.94 × 10⁵ 4.71 × 10⁻² 2.42 × 10⁻⁷ NINA-0444.0128 2.16 × 10⁵ 4.45 × 10⁻² 2.06 × 10⁻⁷ NINA-0445.0128 2.04 × 10⁵ 5.44 × 10⁻² 2.67 × 10⁻⁷ NINA-0456.0128 1.51 × 10⁵ 3.96 × 10⁻² 2.63 × 10⁻⁷ NINA-0460.0128 1.75 × 10⁵ 3.18 × 10⁻² 1.81 × 10⁻⁷ AbN 3.06 × 10⁴ 4.26 × 10⁻² 1.39 × 10⁻⁶ ISTC No binding No binding No binding

Binding Affinity for FX

The anti-FX antibodies analysed showed binding to FX in the affinity range of approximately 0.1 μM to 1.4 μM and fast association (k_(on)) and dissociation (k_(off)) rate for FX. No binding to FX was observed with ISTC.

The anti-FX antibodies analysed similar binding affinity to FX compared to the benchmark antibody AbT.

TABLE E-10-2 Binding affinity and kinetic constants on-rate (kon) and off- rate (koff) of anti-FX antibodies. Anti-FX monospecific antibody nomenclature: TINA-hhhh.llll, wherein hhhh is the numeric identifier of the VH domain (e.g., N0201H) and llll is the numeric identifier of the VL domain (e.g., 0128L). Captured anti-FX antibody k_(on) k_(off) K_(D) (IgG4PE) (1/Ms) (1/s) (M) TINA-0200.0128 (n = 2 1.03 × 10⁵ 1.15 × 10⁻¹ 1.13 × 10⁻⁶ average) TINA-0215.0128 6.84 × 10⁴ 7.87 × 10⁻² 1.15 × 10⁻⁶ TINA-0211.0128 6.57 × 10⁴ 4.47 × 10⁻² 6.80 × 10⁻⁷ TINA-0210.0128 7.64 × 10⁴ 7.44 × 10⁻² 9.74 × 10⁻⁷ TINA-0203.0128 6.80 × 10⁴ 5.98 × 10⁻² 8.80 × 10⁻⁷ TINA-0206.0128 6.72 × 10⁴ 6.67 × 10⁻² 9.92 × 10⁻⁷ TINA-0205.0128 6.26 × 10⁴ 8.53 × 10⁻² 1.36 × 10⁻⁷ TINA-0219.0128 1.01 × 10⁵ 9.11 × 10⁻² 9.05 × 10⁻⁷ TINA-0217.0128 1.02 × 10⁵ 5.74 × 10⁻² 5.64 × 10⁻⁷ TINA-0209.0128 5.90 × 10⁴ 6.79 × 10⁻² 1.15 × 10⁻⁶ TINA-0204.0128 1.09 × 10⁵ 7.85 × 10⁻² 7.18 × 10⁻⁷ TINA-0220.0128 5.38 × 10⁴ 5.60 × 10⁻² 1.04 × 10⁻⁶ TINA-0201.0128 8.67 × 10⁴ 5.02 × 10⁻² 5.79 × 10⁻⁷ TINA-0202.0128 8.87 × 10⁴ 7.20 × 10⁻² 8.12 × 10⁻⁷ TINA-0213.0128 9.69 × 10⁴ 1.33 × 10⁻¹ 1.37 × 10⁻⁶ TINA-0207.0128 1.66 × 10⁵ 1.41 × 10⁻¹ 8.47 × 10⁻⁷ TINA-0214.0128 1.20 × 10⁵ 5.58 × 10⁻² 4.66 × 10⁻⁷ AbN No binding No binding No binding AbT 4.13 × 10⁴ 2.72 × 10⁻² 6.60 × 10⁻⁷ hIgG4PE ISTC No binding No binding No binding

Materials & Methods

SPR was used to determine the binding affinity (K_(D)) to FIX or FX respectively, the kinetic constants on-rate (k_(on)) and off-rate (k_(off)). Analyses was performed using a Biacore 8K (GE Healthcare) system.

Anti-human IgG Fc antibody was immobilised on CM4 chip (GE Healthcare) according to the manufacturer's instructions. The chip surface was activated by amine coupling and subsequently blocked with 1M ethanolamine. The immobilisation run was performed at 25° C. using HBS-EP as immobilisation running buffer.

Monospecific antibodies (referred as ligand) which had been purified on Protein A were captured onto the anti-human IgG Fc CM4 surface at approximately 2 μg/ml. The ligands were injected for 60 seconds at 10 μl/min in all the active channels of all 8 flow channels. The run was performed at 25° C. using neutral pH HBS-P 1X+CaCl₂ 2.5 mM as running buffer.

Human FIX (MW ˜55 KDa) or human FX (MW ˜58 KDa) was reconstituted at 1 mg/ml in the running buffer and used as analyte. The analyte was injected in multiple cycle kinetics (MCK) mode at 3 concentrations (1.5 μM, 500 nM and 166.7 nM) with 120 seconds association phase and 200 seconds (for FIX) or 300 seconds (for FX) dissociation phase, at flow rate 30 μl/sec in both active and reference channels. Three injections of 10 mM Glycine pH 1.5 for 60 sec. at 10 μl/min were used for the regeneration phase.

For the anti-FIX analysis, ISTC antibody hIgG4PE was captured at 1 μg/ml for 60 seconds at 10 μl/min in the reference channel. hIgG4PE ISTC and hIgG1 ISTC were also captured in the active channel as a negative control. The monospecific antibody AbN was used as positive control.

For the anti-FX analysis, the hIgG4PE ISTC was also captured in the active channel as a negative control. The monospecific antibody AbT was used as positive control.

The values for association rate constant (kon), dissociation rate constant (koff) and dissociation constant (K_(D)) were calculated from the binding data by BIAevaluation software. Data were reference and buffer subtracted and fitted into one step biomolecular reaction (Langmuir 1:1) model. The first 30 seconds of dissociation were evaluated in the model.

Example 11. Simultaneous Binding of Bispecific Antibody to FX and FIX

The ability of FIXxFX bispecific antibody IXAX-0436.0202.0128 to bind simultaneously to FIX and FX was demonstrated using SPR. The binding kinetics of the purified bispecific antibody was compared to an isotype control (ISTC). Sensorgrams of the binding indicated that the bispecific antibody bound simultaneously to FIX and FX while no binding to FIX and FX was observed with ISTC. FIG. 21A.

FIX was flown over the surface captured with the bispecific antibody to allow the binding with the first analyte. The interaction between the bispecific antibody generated a baseline response as indicated in the sensogram FIG. 21. The following injection of the second analyte, FX, generated a further increase in signal indicating that FX binds the bispecific antibody already in complex with FIX.

Contrarily no binding to FIX or FX was observed when FIX and FX were flown over the surface where an isotype control was captured, demonstrating the specificity of interaction between FI and FX to the bispecific antibody. FIG. 21A.

Sensorgram for the bispecific antibody can also be compared with sensorgram for monospecific antibody. When the antibody captured is an anti-FX monospecific the same series of injection does not give any significant response when FIX is flown over instead when the second injection is performed (1:1 mixture) approximatively 50 response units (RU) are observed while with the bispecific the response is 25 RU higher. FIG. 21B.

A key feature of the FVII-mimetic bispecific antibody is the ability to bind simultaneously FIX and FX, to promote the conversion of FX into FXa by FIXa. The binding observed represents a biophysical confirmation that the bispecific antibodies described herein can interact simultaneously with Factor IX and Factor IX, which is in agreement with the functional data described in the accompanying Examples.

Materials & Methods

SPR analysis was performed using a Biacore 8K (GE Healthcare) system.

An anti-human IgG Fc antibody was immobilised on CM4 chip (GE Healthcare) according to the manufacturer's instructions. The chip surface was activated by amine coupling and subsequently blocked with 1M ethanolamine. The immobilisation run was performed at 25° C. using HBS-EP as immobilisation running buffer.

Bispecific antibody (ligand), which had been purified by Protein A capture followed by ion exchange chromatography, was captured on to the anti-human IgG Fc CM4 surface at approximately 2 μg/ml. The ligand was injected at 10 μg/ml for 60 seconds at 10 μl/min in one active channel. The run was performed at 25° C. using neutral pH HBS-P 1×+CaCl₂ 2.5 mM as running buffer.

Human FIX and human FX (analytes) were reconstituted at 1.15 mg/ml in the running buffer and used as analytes. Analytes were injected at 10 μM alone or mixed 1:1 (10 μM 10 μM) at 10 μl/min for 180 seconds.

An isotype control hIgG4PE antibody was captured at 10 μg/ml for 60 seconds at 10 μl/min in the reference channel as negative control. A blank injection of buffer was performed for all the samples to be used in the double referencing process. Three injections of 10 mM glycine pH 1.5 for 30 seconds at 30 μl/min were used for the regeneration phase. The data were referenced and buffer subtracted and fitted into Langmuir 1:1 model.

Example 12. Further Optimisation of Anti-FX VH

The anti-FIX binding arm of the bispecific antibody was “fixed” as a VH domain comprising the CDRs of N1280H and a VL domain comprising the CDRs of 0128L, while further refinements were made to the anti-FX VH domain to improve performance. 0128L was used as a common light chain.

Table T identifies mutants of the T0201H VH domain in which one or more residues of the CDRs are mutated to other amino acids. The table shows the name given to each variant VH domain having the identified mutation. In each case, residues other than those indicated are left unchanged. For example, the T0616H VH domain is a Leu11511e mutant of the T0201H VH domain, i.e., in which the leucine (L) at IMGT position 115 in CDR3 is replaced by isoleucine (I). Further residue changes were introduced to the variants containing the single mutations in the T0201H VH domain, resulting in further variants representing combinations of different mutations in the T0201H VH domain. For example, the T0687H VH domain is a Ser111APhe, Cys114Val, Leu115Ile mutant of the T0201H VH domain, i.e., in which the serine at IMGT position 111A in CDR3 is replaced by phenylalanine (T0537H mutation), the cysteine at IMGT position 114 in CDR3 is replaced by valine (T0606H mutation), and the leucine at IMGT position 115 is replaced by isoleucine (T0616H mutation). Sequences of other named anti-FX VH domains can be identified from Table T in the same manner. Refer to FIG. 13 for IMGT numbering.

Bispecific antibodies, purified by Protein A chromatography, were tested for functional activity to look for improvement over the parent bispecific comprising T0201H VH domain.

Improved antibodies were identified in the FXase assay (using Modified FXase Reaction Conditions as detailed in Example 7) and aPTT assay (method as detailed in Example 8).

Mutagenesis of HCDR3 produced improvements in FVIII mimetic activity. HCDRs of VH domains demonstrating improved activity are indicated in FIG. 25. For example, each of the following substitutions and combinations of substitutions in the T0201H VH domain CDR3 was found to improve FVIII mimetic activity (name of resulting VH domain indicated in brackets) (non-exhaustive list):

Gln116Met in CDR3 (T0638H VH);

Leu115Ile in CDR3 (T0616H VH);

Ser111APhe in CDR3 (T0537H VH);

Cys114Ile Leu115Ile (T0666H VH);

Ser111APhe Cys114Ile Leu115Ile (T0678H VH);

Ser111APhe Cys114Leu Leu115Ile (T0681H VH);

Ser111APhe Cys114Val Leu115Ile (T0687H).

Concluding the HCDR3 mutagenesis of T0201H, the VH domains T0687H, T0678H and T0681H demonstrated the strongest activity in the bispecific antibodies.

Functional activity of the bispecific antibodies was still further improved through mutagenesis of HCDR1 and HCDR2 in the anti-FX arm. Starting with T0681H, each amino acid residue of CDR1 and CDR2 was systematically replaced by all other possible amino acids, generating the VH domains numbered T0690H to T0993H identified in Table T.

aPTT and TGA analyses were also conducted to support functional assessment of HCDR1 variants. The VH domains T0736 (S29K mutation), T0713, T0734, T0742, T0774 and T0785 showed improved activity compared with T0681H. Based on the functional analyses of HCDR1 variants of T0681H, VH domain T0736H was selected as the top performer. As compared with T0201H, T0736H combines a Ser29Lys substitution in CDR1 with the Ser111APhe Cys114Val and Leu115Ile substitutions in CDR3.

FXase, aPTT and TGA analyses were also conducted to support functional assessment of HCDR2 variants. Based on the functional analyses of HCDR2 variants of T0681H, the following VH domains were identified to have improved activity compared with T0681H: T0926H (S62K), T850H (I56L), T0925H (S62L), T0951H (G63S), T0958H (S64D), T0989 (T65R) and T0990H (T65S).

Selected CDR1 and CDR2 variants were then combined with selected CDR3, generating further VH domain variants to investigate possible further improvements in activity.

FXase assay data for high-performing antibodies are summaried in FIG. 22 and FIG. 23. The aPTT assay data are summarised in FIG. 24.

Bispecific antibodies comprising the VH domains shown in FIGS. 22 and 23 demonstrated improved or similar clotting times compared with bispecific antibodies comprising T0201H, with T0999H demonstrating the shortest clotting time in aPTT assay.

FXase activity and clotting times were comparable with the comparator bispecific antibody AbE.

FIG. 25 identifies CDRs of VH domains which were progressively improved for FVIII mimetic activity during the mutagenesis process.

Example 13. Strong Activity of Bispecific Antibodies in a Thrombin Generation Assay

The thrombin generation assay (TGA) detects the activation of prothrombin to thrombin in blood plasma. As thrombin is generated it converts a fluorogenic substrate into a fluorophore, which is continuously monitored by a plate reader. The TGA provides a robust measure of the ability of bispecific antibodies to substitute for FVIII in the coagulation cascade in FVIII-deficient plasma, and kinetics of thrombin generation in the TGA are believed to be highly reliable as an indicator of in vivo therapeutic performance of FVIII-mimetic drugs.

Results

To establish a suitable concentration for factor IXa as a TGA trigger, we initially performed TGAs with a fixed concentration of bispecific antibody whilst varying the concentration of FIXa present in the trigger reagent. We determined that a stock solution of 1 ml MP reagent containing 222 nM FIXa is sufficient to trigger thrombin generation for normal pooled human plasma (final concentration, 0.33 nM FIXa) with a Cmax of 418.11 nM thrombin, a Tmax of 7.67 minutes and a lagtime of 5.83 minutes. FIG. 26. These values are comparable with the reference range in healthy adults (see, e.g., Table 3 of ref [11]) and validate the use of FIXa as a TGA trigger.

Bispecific antibody VH domain T0201H and CDR1, CDR2 and CDR3 combinatorial variants of T0201H were expressed with FIX N1280H arm and N0128 common light chain in HEK cells, purified by protein A chromatography and analysed at a final concentration of 133 nM and 80 nM. The VH domain variants exhibited shortening lagtime, increasing Cmax and shorter time to peak compared with T0201H, with T0999 demonstrating the largest thrombin peak height and shortest time to peak at both concentrations analysed. Performance of at least IXAX-1280.0999.0128 was comparable with that of AbE and of the emicizumab calibrator. AbE demonstrated a lagtime, peak height and time to peak of 2.5 mins, 291.8 nM and 6.0 minutes respectively, and IXAX-1280.0999.0128 demonstrated a lagtime, peak height and time to peak of 2.0 mins, 317.2 nM and 5 minutes. FIG. 27. FIG. 28. As illustrated in FIG. 27 and in order of increasing Tmax, we observed a Tmax of 5, 6.83, 6.83, 7, 7.67, 13.17, 15 and 15.67 minutes for bispecific antibodies comprising T0999, T0687, T0736, T0678, T0681, T0666, T0201 and T0596 respectively.

TABLE E13-1 Recorded parameters from TGA carried out on FVIII deficient plasma spiked with CDR1, CDR2 and CDR3 single and combinatorial mutants of T0201H in bispecific antibody IXAX- 1280.0201.0128 at final concentration of 133 nM. ETP = endogenous thrombin potential. Isotype VH T0201 T0596 T0666 T0678 T0681 T0687 T0736 T0999 AbE Control Lagtime (min) 5.0 5.2 4.5 2.7 3.0 2.7 2.7 2.0 2.5 18.0 ETP (nmol/L 1774.4 1724.2 1883.6 2053.5 1932.4 2077.5 1805.4 2099.8 1951.9 −1.0 thrombin × min) Maximal Peak 134.9 126.4 160.4 259.6 235.6 267.8 237.5 317.2 291.8 19.9 Height (nM) Time to peak 15.0 15.7 13.2 7.0 7.7 6.8 6.8 5.0 6.0 38.3 (min) Velocity Index 13.5 12.0 18.5 59.9 50.5 64.4 57.1 105.7 83.6 1.0 (nM/min) Tail Start (min) 39.0 39.8 37.0 29.8 30.2 29.5 28.2 26.7 27.3 −1.0

TABLE E13-2 Recorded parameters from TGA carried out on FVIII deficient plasma spiked with CDR1, CDR2 and CDR3 single and combinatorial mutants of T0201H in bispecific antibody IXAX-1280.0201.0128 at final concentration of 80 nM. ETP = endogenous thrombin potential. Refer to FIG. 27. Isotype VH T0201 T0596 T0666 T0678 T0681 T0687 T0736 T0999 AbE Control Lagtime (min) 5.0 5.3 4.7 2.7 3.0 2.7 2.7 2.0 3.0 19.0 ETP (nmol/L 1766.2 1793.8 1757.9 1975.4 1891.7 1927.1 1933.3 1986.1 1878.6 −1.0 thrombin × min) Maximal Peak 126.6 125.9 148.2 269.7 231.4 266.2 248.9 308.0 255.8 19.7 Height (nM) Time to peak 15.7 16.0 13.5 6.7 7.8 6.7 6.7 4.8 7.2 39.0 (min) Velocity Index 11.9 11.8 16.8 67.4 47.9 66.5 62.2 109.2 61.6 1.0 (nM/min) Tail Start (min) 41.0 41.5 37.0 28.7 29.8 29.5 30.0 26.8 29.8 −1.0

TABLE E13-2 Recorded parameters from TGA carried out with (i) emicizumab calibrator and (ii) normal pooled plasma spiked with PBS. The emicizumab calibrator is originally 100 ug/ml = 0.1 mg/ml = 666.666 nM. Dilution 125/80 = 1.5625 (80 plasma, 20 Calibrator/MP, 20 FluCa, 5 PBS spike) provides final emicizumab concentration of 426.6 nM for the calibrator. Refer to FIG. 27. Calibrator Normal Plasma Lagtime (min) 2.0 4.8 Endogenous Thrombin Potential 1538.0 1425.7 (ETP) (nmol/L thrombin × min) Maximal Peak Height (nM) 308.6 371.2 Time to peak (min) 4.5 6.5 Velocity Index (nM/min) 124.1 222.7 Tail Start (min) 23.7 22.7

For a dose response TGA, bispecific antibody IXAX-1280.0999.0128 was expressed in HEK cells, purified by Protein A chromatography and bispecific heterodimer purified by ion exchange chromatography. Using 0.3 nM FIXa trigger, dose response of Cmax (nM) and Tmax (min) in the TGA was carried out on FVIII deficient plasma spiked with bispecific antibody IXAX-1280.0999.0128 and compared against emicizumab calibrator. Both bispecific antibodies demonstrated a linear decrease in Cmax with increasing antibody concentration. IXAX-1280.0999.0128 achieved a greater Cmax than the calibrator antibody, this increase being more pronounced at lower bispecific antibody concentrations. See FIG. 29.

TABLE E13-3 Recorded parameters from TGA carried out on FVIII deficient plasma spiked with CDR1, CDR2 and CDR3 single and combinatorial mutants of T0201H in bispecific antibody IXAX-1280.0201.0128. Refer to FIG. 29. IXAX-1280.0999.0128 Dose Response FIxa Trigger (0.3 nM) Normal Isotype 300 nM 100 nM 30 nM 10 nM 3 nM 1 nM Plasma Control Lagtime (min) 2 1.83 2.17 3.33 4.5 5.83 6.17 18 Endogenous 2703 2170 2266 2311 2208 2088 1629 −1 Thrombin Potential (ETP) (nmol/L thrombin × min) Maximal Peak 377.14 348.38 327.7 274.76 208.45 124.11 466.31 23.47 Height (nM) Time to peak 5.33 4.33 5.5 8 12 18.83 7.83 39.17 (min) Velocity Index 113.14 140.01 98.31 58.88 27.82 9.62 279.78 1.11 (nM/min) Tail Start (min) 28.67 26.83 28.67 31.5 35.33 44.5 23.33 −1

TABLE E13-4 Recorded parameters from TGA carried out with emicizumab calibrator. Refer to FIG. 29 Emicizumab Calibrator Dose Response FIxa Trigger (0.3 nM) Normal Isotype 300 nM 100 nM 30 nM 10 nM 3 nM 1 nM Plasma Control Lagtime (min) 2 2.33 3 4.33 5.5 6.67 6.17 18 Endogenous 1884 2363 2075 1733 1524 1634 1629 −1 Thrombin Potential (ETP) (nmol/L thrombin × min) Maximal Peak 343.94 351.58 255.87 152.08 89.33 67.38 466.31 23.47 Height (nM) Time to peak 4.17 5.5 8.17 12.5 18.33 23 7.83 39.17 (min) Velocity Index 160.26 111.69 50.03 18.67 6.96 4.13 279.78 1.11 (nM/min) Tail Start (min) 25 27.67 29.33 35.33 43.33 58.17 23.33 −1

Materials & Methods

For the initial experimental work to establish a suitable concentration of factor IXa as a TGA trigger, 80 μl normal pooled plasma, taken from healthy individuals (Helena Biosciences), was mixed with 20 μl of trigger reagent (Microparticle (MP) reagent which is composed of phospholipids only containing varying amounts of FIXa) in Immulon 2HB transparent U-bottom 96 well plates (ThermoFisher #3665). All reagents were used according to manufacturer's instructions, pre-warmed to 37° C. in a water bath.

Once a final concentration of 0.33 nM FIXa was determined to be sufficient to trigger thrombin generation for normal plasma, the same assay conditions including 0.3 nM FIXa were applied with FVIII-depleted plasma in calibrated automated thrombogram assays.

FVIII immunodepleted plasma (Helena Biosciences) was mixed with 20 μl of trigger reagent (Microparticle (MP) reagent which is composed of phospholipids only containing 222 nM FIXa, final concentration 0.33 nM) in Immulon 2HB transparent U-bottom 96 well plates. All reagents were used according to manufacturer's instructions, pre-warmed to 37° C. in a water bath. A TGA dose response was carried out starting at 300 nM of test bispecific antibody or of emicizumab calibrator ((emicizumab spiked into FVIII deficient plasma (Enzyme Research Laboratories)) with a 1 in 3 dilution series over five points. A human IgG4 isotype control antibody was used as negative control, and normal (FVIII+ve) pooled plasma spiked with PBS was used as positive control.

Samples were measured in duplicate, accompanied by duplicate calibrator wells containing a thrombin calibrator (containing a pre-determined quantity of thrombin) in the same plasma. The 96 well plate was warmed to 37° C. in a Fluoroskan Ascent plate reader (Thermo) for 10 minutes. Thrombin generation commenced upon addition of 20 μl FluCa reagent (fluorogenic substrate, ZGGR-AMC (2.5 mM), in buffer containing 100 mM CaCl₂). TGA reagents were obtained from Stago. Increase in fluorescence over time was monitored by the plate reader.

A thrombin calibrator curve was run alongside each sample being investigated. Using a calibrator, with a known concentration of thrombin, the amount of thrombin generated in a sample under investigation can be calculated from the fluorescent signal obtained using software ThrombinoscopeBV. Fluorescence from test wells was calibrated against fluorescence from the thrombin calibrator wells, to determine the equivalent thrombin generated in the test wells.

Run data were analysed using Stago analysis software. The amount of thrombin generated was determined using the thrombin calibrator curve with known activity. The following aspects of the thrombogram were determined: lag time (minutes), endogenous thrombin potential (ETP; area under the thrombogram, nM thrombin/minute), peak height (Cmax; nM thrombin), time to peak (Tmax/minutes), velocity index (VI; nM/minute, slope between lag time and time to peak) and tail start (minutes; time at which the thrombin generation has come to an end).

Example 14. Dose Response and Potency in Thrombin Generation Assay

To evaluate the maximal thrombin peak height (Cmax, nM Thrombin) and time to peak (Tmax, minutes) of bispecific antibody IXAX-1280.0999.0325 we performed thrombin generation assays (TGA) in human FVIII-depleted plasma using a full antibody concentration dose response according to the method set out in Example 13. Data generated from dose response curves was fitted using a non-linear log[antibody] vs response parameter variable slope model (4 parameter logistic regression model). AbE was included for comparison. IXAX-1280.0999.0325 and AbE used in this assay were determined by mass spectrometry to be close to 100% heterodimer, with no homodimeric contaminants detected.

Over a prospective therapeutic window spanning 300 to 30 nM, equivalent to 45 to 4.5 μg/ml, we observed equivalent (within 10%) or greater Cmax (nM Thrombin) values for IXAX-1280.0999.0325 compared to AbE at all concentrations analysed (FIG. 30). Cmax of IXAX-1280.0999.0325 was close to that of a normal plasma control when the concentrations of the bispecific antibody were between 100 and 300 nM (FIG. 30 and FIG. 31). In contrast, Cmax of AbE only reached the normal level when IgG concentration is 300 nM. In this study, the EC50 of IXAX-1280.0999.0325 (8.0 nM) was approximately 15% of the EC50 of AbE (54.4 nM).

Using the Cmax curve, it can be predicted that IXAX-1280.0999.0325 can achieve the same activity as 45 μg/mL of emicizumab when its concentration is equal to or greater than 8 μg/mL, which suggests a potential efficacy advantage with IXAX-1280.0999.0325 compared with emicizumab.

Analysis of the same dose response but with respect to Tmax, we observed equivalent (within 10%) or less than (or reduced) Tmax values for IXAX-1280.0999.0325 compared to AbE at all concentrations analysed (FIG. 32). Calculated EC50 values based on Tmax values obtained are 1.65 nM for IXAX-1280.0999.0325 (circle) compared to 2.8 nM for AbE (square).

In respect to the therapeutic ranges indicated in FIGS. 30 and 32, we observe with IXAX-1280.0999.0325 Cmax and Tmax dose response curves which are greater and lower compared to AbE, respectively.

TABLE E14-1 Non-linear fit of Cmax. Best fit values for log of antibody concentration vs Cmax. Variable slope (4 parameters). IXAX-1280.0999.0325 Ab_E Bottom 18.20 4.053 Top 392.0 399.9 LogEC50 −8.098 −7.264 HillSlope 1.111 1.135 EC50 7.984e−009 5.440e−008 Span 373.8 395.8

TABLE E14-2 Non-linear fit of Tmax. Best fit values for log of antibody concentration vs Cmax. Variable slope (4 parameters). IXAX-1280.0999.0325 Ab_E Bottom 2.888 −0.2454 Top 37.14 62.16 LogEC50 −8.781 −8.548 HillSlope −0.7713 −0.5577 EC50 1.654e−009 2.829e−009 Span 34.25 62.41

The activities of three further bispecific antibodies (BiAb 2, 3 and 4) were also assessed in the TGA and compared against the performance of IXAX-1280.0999.0325 (BiAb 1) and commercially available emicizumab calibrator (Enzyme Research Laboratories) in commercially available human FVIII-depleted plasma (Helena Biosciences). BiAbs were as follows, each including heavy chain constant regions SEQ ID NO: 409 and SEQ ID NO: 410 respectively in the two heavy chains, and lambda light chain constant region SEQ ID NO: 146 in the common light chain:

-   1. IXAX-1280.0999.0325. Anti-FIX heavy chain SEQ ID NO: 419, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414. -   2. IXAX-1454.0999.0325. Anti-FIX heavy chain SEQ ID NO: 424, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414. -   3. IXAX-1441.0999.0325. Anti-FIX heavy chain SEQ ID NO: 426, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414. -   4. IXAX-1442.0736.0325. Anti-FIX heavy chain SEQ ID NO: 428, anti-FX     heavy chain SEQ ID NO: 430, common light chain SEQ ID NO: 414.

BiAb_1, 2, 3 and 4 dose-dependently increased thrombin peak height (Cmax), and dose-dependently decreased time to peak (Tmax) in the same manner as emicizumab. The top of Cmax curve of BiAb_1 was measured at about 368 nM, higher than that of emicizumab (334.8 nM). EC50 (Cmax) of BiAb_1, 2, 3, and 4 were similar to each other and had calculated EC50s of 6.45 nM, 5.87 nM, 5.2 nM and 4.81 nM respectively, representing EC50s between 26% and 35% of the EC50 of emicizumab (18.33 nM). FIG. 33 A. Calculated EC50 (Tmax) values for BiAb_1 to BiAb_4 were 0.56 nM, 0.65 nM, 1.08 nM and 0.93 nM respectively, compared with 2.53 nM for emicizumab. FIG. 33 B.

In a third study, BiAb_1 (IXAX-1280.0999.0325) was again compared with commercially available emicizumab calibrator by using TGA assay in human FVIII-depleted plasma. BiAb_1 dose-dependently increased thrombin peak height (Cmax), and dose-dependently decreased time to peak (Tmax). FIG. 34. The top of Cmax curve for BiAb_1 was about 396.5 nM, higher than that of emicizumab (286.3 nM). EC50 of BiAb_1 was 7.7 nM, approximately 30% of the EC50 of emicizumab (25.9 nM).

Assay to assay variation is observed between the TGA as shown in FIG. 34, FIG. 33 and FIG. 30, in which BiAb_1 exhibited Cmax of approximately 400 nM, 375 nM and 375 nM respectively and in which emicizumab exhibited Cmax of 275 nM, 300 nM and 350 nM. Despite variation in the absolute readings, the trends observed were the same in each instance of the TGA.

In summary, TGA data with either the commercially available emicizumab calibrator or the generated reference antibody AbE consistently indicated an efficacy advantage for BiAb_1 (IXAX-1280.0999.0325) compared with emicizumab. An advantage was also observed with the other antibodies tested (BiAb_2, BiAb_3 and BiAb_4).

According to an FDA multi-disciplinary review of emicizumab, a median annualized bleeding rate (ABR) of 0 would be achieved at emicizumab steady state trough plasma concentration≥45 μg/mL[17]. Using the Cmax curves from the TGA described above, it is predicted that BiAb_1 can achieve the same activity as 45 μg/mL of emicizumab when its concentration is equal to or greater than about 2-4 μg/mL. This observation suggests a potential efficacy and/or dosing advantage with respect to emicizumab. The differences in activity potentially mean that the bispecific antibody can achieve the same therapeutic effect when administered at lower dose and/or less frequently than emicizumab, representing a clinical advantage. Although the higher Cmax indicates the potential for a more powerful procoagulant capability, the magnitude of this increase is unlikely to be associated with safety concerns.

Example 15. Affinities of Optimised Antibody Arms

Affinity and kinetics of purified anti-FIX and anti-FX antibodies for binding to their respective antigens was determined by SPR as described in Example 10 above.

The anti-FIX antibodies showed binding to FIX with an affinity range of approximately 0.05 μM−0.3 μM (50-300 nM), with a general trend of increasing affinity (lower K_(D)) and faster off-rate correlating with greater activity in the bispecific antibody. Table E15-1.

TABLE E15-1 Captured anti-FIX IgG k_(on) (1/MS) k_(off) (1/s) K_(D) (M) Isotype control antibody No binding No binding No binding AbN 3.64 × 10⁴ 6.48 × 10⁻² 1.78 × 10⁻⁶ NINA-0128 1.37 × 10⁵ 4.92 × 10⁻² 3.59 × 10⁻⁷ NINA-0436.0128 1.46 × 10⁵ 5.23 × 10⁻² 3.59 × 10⁻⁷ NINA-0511.0128 1.34 × 10⁵ 1.75 × 10⁻² 1.31 × 10⁻⁷ NINA-1091.0128 1.61 × 10⁵ 3.52 × 10⁻² 2.18 × 10⁻⁷ NINA-1172.0128 2.25 × 10⁵ 1.71 × 10⁻² 7.64 × 10⁻⁸ NINA-1280.0128 1.92 × 10⁵ 1.00 × 10⁻² 5.23 × 10⁻⁸

The anti-FX antibodies showed binding to FX with an affinity range of approximately 0.3-3 μM. Table E15-2. Anti-FX antibody MONA was included as a control low affinity antibody with VH and VL domains from an IgM clone obtained from the single cell sorting (Example 1).

TABLE E15-2 Captured anti-FX IgG k_(on) (1/MS) k_(off) (1/s) K_(D) (M) Isotype control antibody No binding No binding No binding AbT 2.50 × 10⁴ 3.85 × 10⁻² 1.54 × 10⁻⁶ TINA-0200.0128 4.43 × 10⁴ 1.12 × 10⁻¹ 2.52 × 10⁻⁶ TINA-0201.0128 5.90 × 10⁴ 4.82 × 10⁻² 8.16 × 10⁻⁷ TINA-0202.0128 5.13 × 10⁴ 7.09 × 10⁻² 1.38 × 10⁻⁶ TINA-0616.0128 4.19 × 10⁴ 2.22 × 10⁻² 5.30 × 10⁻⁷ TINA-0638.0128 6.99 × 10⁴ 2.32 × 10⁻² 3.33 × 10⁻⁷ TINA-0666.0128 4.49 × 10⁴ 4.17 × 10⁻² 9.27 × 10⁻⁷ MONA_IgG4PE 5.36 × 10⁴ 5.24 × 10⁻² 9.78 × 10⁻⁷

Example 16. Initial Biophysical Assessment

To evaluate expression of the bispecific antibodies, IXAX-1172.0201.0128 was chosen as a representative antibody for minipool analysis. Minipool analysis allows screening of CHO stably transfected cells expressing large amounts (at least 1 g/l) of heterodimeric bispecific antibody and represents a means of evaluating stable bispecific antibody expression.

Using standard Lonza fed-batch overgrowth protocols for stably transfected CHO-K1 cells, bispecific antibodies were expressed. After transfection, 5000 viable cells were aliquoted per well to generate multiple minipools. 8 were taken forward based on antibody titres as measured by Octet.

Cells were harvested, filtered and purified by Protein A chromatography to isolate the antibodies from the supernatant. Antibody concentration (mg) was quantified by OD280, total amount of antibody (mg) was calculated accordingly based on volume of sample and a purification yield (mg/L) assigned according to cell culture volume. The relative percentages of heterodimer and homodimers in each of the 8 minipool samples was determined using imaged capillary isoelectic focusing (icIEF) (Protein Simple, Maurice). Homodimer and heterodimer peaks were assigned using transiently expressed reference homodimer arms for FIX and FX.

We were able to isolate stably transfected cells expressing approximately 1 g/L bispecific antibody with up to approximately 95% heterodimer (e.g., as shown for MP_1 and MP_7, FIG. 35).

Bispecific antibody activity in FXase assay correlated with % heterodimer with a Pearson's correlation coefficient of 0.99 (FIG. 36).

Example 17. Purification of Bispecific Antibody

Co-expression of the two heavy chains and one common light chain of a bispecific antibody generates a composition comprising the bispecific antibody plus monospecific antibody byproducts. These may be separated by ion exchange chromatography, exploiting differences in the isoelectric point of the bispecific heterodimer compared with the monospecific homodimers.

Bispecific antibody IXAX-1280.0999.0128 comprises anti-FIX heavy chain SEQ ID NO: 419, anti-FX heavy chain SEQ ID NO: 421 and common light chain SEQ ID NO: 405. The bispecific antibody was purified following co-expression of these polypeptides in HEK cells, using protein A chromotography to isolate the antibodies from cell supernatant, followed by ion exchange chromatography to isolate the heterodimer.

Bispecific antibody IXAX-0436.0201.0128 comprises anti-FIX heavy chain comprising VH domain SEQ ID NO: 324 and an IgG4 human heavy chain constant region with P (hinge) mutation and K439E, anti-FX heavy chain comprising VH domain SEQ ID NO: 470 and IgG4 human heavy chain constant region with P (hinge) mutation and E356K, and common light chain SEQ ID NO: 405.

Bispecific antibody IXAX-0436.0202.0128 comprises anti-FIX heavy chain comprising VH domain SEQ ID NO: 324 and an IgG4 human heavy chain constant region with P (hinge) mutation and K439E, anti-FX heavy chain comprising VH domain SEQ ID NO: 472 and IgG4 human heavy chain constant region with P (hinge) mutation and E356K, and common light chain SEQ ID NO: 405.

Bispecific antibody IXAX-1172.0201.0128 comprises anti-FIX heavy chain comprising VH domain SEQ ID NO: 440 and an IgG4 human heavy chain constant region with P (hinge) mutation and K439E, anti-FX heavy chain comprising VH domain SEQ ID NO: 470 and an IgG4 human heavy chain constant region with P (hinge) mutation and E356K, and common light chain SEQ ID NO: 405.

Ion exchange chromatography cleanly separated each antibody composition into its component parts. Baseline separation was observed. Anti-FIXxFX heterodimeric bispecific antibody is separated from homodimeric contaminant anti-FIX and/or anti-FX monospecific antibodies.

FIG. 37a shows successful purification of IXAX-1280.0999.0128 from a composition comprising the bispecific antibody mixed with anti-FIX homodimer NINA-1280.0128 and anti-FX homodimer TINA-0999.0128.

FIG. 37b shows successful purification of IXAX-0436.0202.0128 from a composition comprising the bispecific antibody mixed with anti-FIX homodimer NINA-0436.0128 and anti-FX homodimer TINA-0202.0128. The chromatogram represents cation ion exchange purification of N436 bispecific antibody from homodimer contaminants using a series of stepwise elutions using increasing concentrations of NaCl up to 500 nM in Sodium acetate pH 5. Peak 1 represents anti-FIX homodimer antibody; peak 2, anti-FIX/FX bispecific antibody and peak 3 represents anti-FX homodimer antibody. Peak 1, 2 and 3 make up 18%, 79% and 3% total peak area respectively.

FIG. 37c shows successful purification of IXAX-1172.0201.0128 from a composition comprising the bispecific antibody and anti-FIX homodimer NINA-1172.0128. The column purification here yielded 31.5% anti-FIX homodimer and 68.5% bispecific heterodimer. The chromatogram represents cation ion exchange purification of N1172 bispecific antibody from anti-FIX homodimer contaminants using an initial stepwise elution to remove weakly bound Peak 1 (anti-FIX homodimer) followed by a gradient elution using increasing concentrations of NaCl to elute the anti-FIX/FX bispecific. The presence of anti-FX homodimer was not detected.

Materials & Methods

For IXAX-1280.0999.0128 purification, bispecific antibody was transiently expressed in Expi293F HEK cells. Cell culture supernatant was harvested, filtered and loaded on to a 5 ml HiTrap MabSelect Sure (MSS) column (GE Healthcare) equilibrated with 1× phosphate buffered saline (PBS). The column was washed with 5 column volumes of PBS and bound antibody was eluted using IgG elute (ThermoFisher). Eluted bispecific antibody was dialysed into 1×PBS overnight at 4° C. and concentrated using a centrifugal filter unit with a 10 kDa molecular weight cut off.

Chromatography was performed at room temperature. A 1 ml HiTrap Capto SP column (GE Healthcare) was equilibrated with 20 mM sodium phosphate, pH 6.0 and 0.5 mg of Protein A purified material, diluted 1:20 in equilibration buffer (20 mM sodium phosphate, pH 6.0), was loaded on to the column. The column was subsequently washed with 10 column volumes of equilibration buffer followed by a linear gradient (100% B over 90 column volumes to 500 mM NaCl) to elute the bispecific antibody and monospecific contaminants. In this process, buffer is progressively changed from A (20 mM sodium phosphate, pH 6.0, no salt) to B (buffer A with the addition of 500 mM NaCl) over 90 cv at a flow rate of 1 ml/min for the 1 ml column.

For IXAX-0436.0202.0128 purification, a stepwise gradient including washes at three different ionic strengths was applied using varied proportions of Buffer A (50 mM sodium acetate, pH 5) and Buffer B (50 nM sodium acetate and 500 mM sodium chloride).

For IXAX-1172.0201.0128 purification. an initial stepwise elution was used to remove weakly bound Peak 1 (anti-FIX homodimer) followed by a gradient elution using increasing concentrations of NaCl to elute the anti-FIX/FX bispecific.

Subsequently, the following bispecific antibodies were expressed in CHO cells:

-   5. IXAX-1280.0999.0325. Anti-FIX heavy chain SEQ ID NO: 419, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414. -   6. IXAX-1454.0999.0325. Anti-FIX heavy chain SEQ ID NO: 424, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414. -   7. IXAX-1441.0999.0325. Anti-FIX heavy chain SEQ ID NO: 426, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414. -   8. IXAX-1442.0736.0325. Anti-FIX heavy chain SEQ ID NO: 428, anti-FX     heavy chain SEQ ID NO: 430, common light chain SEQ ID NO: 414. -   9. IXAX-1442.0687.0325. Anti-FIX heavy chain SEQ ID NO: 428, anti-FX     heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.

Each of these bispecific antibodies includes heavy chain constant regions SEQ ID NO: 409 and SEQ ID NO: 410 respectively for the two heavy chains, and lambda light chain constant region SEQ ID NO: 146 in the common light chain.

The titres observed from transient expression of each of antibodies 1 to 5 above in CHO cells were comparable to titres for a monospecific isotype control antibody. Stable pools and mini-pools (up to 4,000 cells seeded after transfection) were also generated. Although the stable pools produced low percentages (11-19%) of heterodimeric antibody, mini-pools with titres up to 4.9 g/I and percentages of heterodimers up to 82% were established from a limited number of screened mini-pools.

After protein A purification, cation exchange chromatography was used to remove homodimeric by-products to generate high-purity materials suitable for use in functional assays and for developability screening.

Using a gradient cation exchange method, antibodies 1 to 4 were separated from the homodimeric by-products, providing 91-96% heterodimer in the eluted material. Thus, even with this preliminary purification method we were able to obtain 91-96% pure heterodimer. FIG. 38. No comparable homodimer/heterodimer separation was observed for antibody 5 using this technique.

Example 18. Quality Assessment by Mass Spectrometry

The structural integrity of therapeutic monoclonal antibodies can be compromised by multiple types of post-translational modifications which result in product heterogeneity. Mass spectrometry (MS) was used to characterize and evaluate the quality of the bispecific antibodies after cation exchange purification.

After cation exchange separation as described in Example 17, the three different species (anti-FIX/anti-FX heterodimer, anti-FIX homodimer and anti-FX homodimer respectively) in the eluted composition of BiAb_1 IXAX-1280.0999.0325 were analysed by MS. Molecular weights (MW) of the three molecules determined by MS matched the theoretical MW predicted by amino acid sequences of BiAb_1. MS results thus confirmed the identity and the purity of FIX/FX heterodimer after cation exchange purification.

Example 19. Stability Assessment

After purification as described in Example 17, BiAb_1, 2, 3 and 4 respectively were buffer exchanged to either buffer 1 (sodium acetate, pH 5.5) or buffer 2 (citrate/phosphate, pH 6.0), stored for 2 weeks at 4° C., for 4 weeks at 25° C., or underwent 1× freeze/thaw cycle. The concentration of IgG was measured before and after treatment to calculate the loss of antibodies due to the treatment. SEC-HPLC was also performed before and after treatment to monitor for bispecific antibody degradation and aggregation. No obvious loss or degradation of BiAb_1, 2, 3 or 4 was observed. These four bispecific antibodies were thus all stable in both buffer 1 and buffer 2.

Example 20. Dose Response and Potency in FXase Assay

A FXase kinetic assay was conducted to measure the factor VIII mimetic activity of IXAX-1280.0999.0325 and AbE in a dose response to determine their EC50 values as per Example 7. Data generated from dose response curves was fitted using a non-linear log[antibody] vs response parameter variable slope model (4 parameter logistic regression model). FIG. 39. The y-axis are plotted OD405 nm values at a 600 second assay timepoint. The EC50 values for both IXAX-1280.0999.0325 and AbE were calculated from a non-linear regression curve of the data, giving an EC50 of 3.99 nM for IXAX-1280.0999.0325 and EC50 of 261.2 nM for AbE. These are approximations since the dose response curve was incomplete. Despite this it is evident that IXAX-1280.0999.0325 has a significantly lower EC50 than AbE. The activity of IXAX-1280.0999.0325 and AbE is dose dependent. IXAX-1280.0999.0325 demonstrated higher FVIII mimetic activity, in particular at lower concentrations, compared with AbE.

TABLE E20-1 Kinetic FXase best fit values for log of antibody concentration vs response. Variable slope (4 parameters). IXAX-1280.0999.0325 Ab_E Bottom 0.1620 0.1103 Top 0.6872 0.9858 LogEC50 −8.399 −6.583 HillSlope 2.707 1.087 EC50 3.993e−009 2.612e−007 Span 0.5252 0.8756

Example 21. Hyphen Assay Dose Response

A chromogenic assay (HYPHEN BioMed), which analyses factor Xa production in human plasma, was used to measure the factor VIII mimetic activity of IXAX-1280.0999.0325 and AbE. In this assay, FXa generation is proportional to the OD405 measured after chromogenic substrate addition. Antibody concentration dose response curves were generated and fitted using a non-linear log[antibody] vs response parameter variable slope model (4 parameter logistic regression model). EC50 values based on the dose responses were calculated. An EC50 value of 5.92 nM was calculated for IXAX-1280.0999.0325 compared with 15.43 nM for AbE.

IXAX-1280.0999.0325 consistently achieved greater FVIII mimetic ability than AbE over almost all concentrations. At 60 nM the A405 nm had saturated at 4.988 for both molecules. IXAX-1280.0999.0325 retained this saturation at 20 nM while AbE presented with a decreased A405 nm of 3.457. At the lowest concentration of 0.028 nM, IXAX-1280.0999.0325 displayed over 4-fold greater absorbance than AbE. The calculated EC50 values confirm that IXAX-1280.0999.0325 shows a superior potency when comparing to AbE across a dose response assay. FIG. 40.

TABLE E21-1 Hyphen FXase EC50. Best fit values for log of antibody concentration vs response. Variable slope (4 parameters). IXAX-1280.0999.0325 Ab_E Bottom 0.7007 0.2581 Top 5.195 5.861 LogEC50 −8.227 −7.812 HillSlope 1.848 1.231 EC50 5.924e−009 1.543e−008 Span 4.495 5.603

Materials & Methods

The BIOPHEN FVIII:C (Ref. 221402) kit was used following manufacture's assay protocol. Briefly, FVIII deficient plasma (Helena Biosciences Europe) was diluted 1:40 using Tris-BSA buffer (R4) and 45 μl was added to a clear bottom 96-well plate. 5 μl of bispecific antibody was added to the diluted plasma. 50 μl each of reagent R1 (FX) and R2 (FIXa), pre-incubated to 37° C., was added to each well and incubated at 37° C. for five minutes. Subsequently, 50 μl of reagent R3 (SXa-11, chromogenic reagent) was added, mixed and incubated for an additional five minutes, exactly. Addition of 50 μl 20% acetic acid terminated the reaction. Generation of Factor Xa was monitored through the ability of factor Xa to cleave a specific factor Xa substrate (SXa-11). Cleavage of this substrate releases the coloured product, pNA, which can be monitored using a spectrophotometer at 405 nM and compared to a blank sample.

IXAX-1280.0999.0325 and AbE used in this assay were determined by mass spectrometry to be close to 100% heterodimer, with no (or low levels of) homodimeric contaminants detected.

IXAX-1280.0999.0325 and AbE samples were diluted using a 1:3 dilution series with PBS as diluent. 5 μL volume was added to 45 μL factor VIII deficient plasma. The final concentrations (nM) of each sample in the dilution series (when assayed) were: 60.0, 20.0, 6.67, 2.22, 0.741, 0.247, 0.082, and 0.028. Concentrations were converted to log(M) and plotted. A non-linear regression was plotted on the graph to enable EC50 calculation.

Example 22. Dose Response and Potency in Plasma Coagulation Assay

We evaluated the activated partial thromboplastin time (aPTT) of bispecific antibody AbE against IXAX-1280.0999.0325 using a full antibody concentration dose response (method according to Example 8). Data generated from dose response curves were fitted using a non-linear log[antibody] vs response parameter variable slope model (4 parameter logistic regression model).

Over the concentration values analysed we observed equivalent aPTT values (within 10%) for IXAX-1280.0999.0325 compared to AbE at all concentrations analysed (FIG. 41). Dose response curves were compared to a human IgG4 monoclonal antibody isotype control, which demonstrated no haemostatic efficacy. Calculated EC50 values based on aPTT values obtained were 2.1 nM for IXAX-1280.0999.0325 (circle) compared with 2.0 nM for AbE (square).

With respect to a prospective therapeutic range of 30-300 nM, we observe with IXAX-1280.0999.0325 an aPTT dose response curve equivalent to that of AbE.

IXAX-1280.0999.0325 and AbE used in this assay were determined by mass spectrometry to be 100% heterodimer, with no homodimeric contaminants detected.

Example 23. Activity in the Presence of Anti-FVIII Inhibitory Antibodies

Factor VIII replacement therapy can become ineffective for treating patients with haemophilia A if the patient develops alloantibodies against the exogenously administered FVIII. Inhibitory anti-FVIII alloantibodies may block the binding of FIX, phospholipid and von Willebrand factor to FVIII, rendering it inactive.

Advantageously, therapeutic bispecific antibodies are insensitive to the presence of FVIII alloantibodies in a patient's blood, as the alloantibodies have specificity to FVIII. This is confirmed by the ability of a bispecific antibody to functionally restore haemostasis in plasma taken from an inhibitor patient. In this Example, we demonstrate this using two haemostatic assays: activated partial thromboplastin time (aPTT) and Thrombin Generation Assay (TGA) using plasma from a patient with haemophilia A having inhibitory alloantibodies (referred to as “inhibitor plasma”). A restoration of clotting time indicated that the bispecific antibodies analysed are functional in the presence of a FVIII inhibitory alloantibody. Thus, the data presented here indicate that IXAX-1280.0999.0325 and IXAX-1441.0999.0325 will be able to functionally rescue clotting time in patients who have inhibitory alloantibodies against FVIII.

The Bethesda assay or the Nijmegen-Modified Bethesda assay is used measure the titre of alloantibodies against FVIII. In these assays, different dilutions of patient's plasma are mixed with an equal volume of ‘normal’ plasma and left to incubate for a period of time and the level of FVIII is measured. Presence of an inhibitor is indicated when a decrease in residual FVIII is observed. The unit of measurement in these assays are known as Bethesda Units (BU)—a higher BU indicating greater inhibition and lower residual FVIII activity. The experiments described here used patient plasma having a specific inhibitor level of 70 BU.

aPTT

IgG4 bispecific antibodies IXAX-1280.0999.0325, IXAX-1441.0999.0325 and AbE were expressed in CHO cells then purified by Protein A chromatography followed by cation exchange chromatography to separate active heterodimer from contaminating homodimers and analysed at six different concentrations 300, 100, 33.3, 11.1, 3.7 and 1.23 nM by aPTT. aPTT was carried out as per Example 8. Data generated from dose response curves were fitted using a non-linear log[antibody] vs response parameter variable slope model (4 parameter logistic regression model). Both IXAX-1280.0999.0325 and IXAX-1441.0999.0325 IgG4 antibodies were able to rescue the clotting defect in the inhibitor patient sample in a similar manner to AbE. FIG. 42.

Thrombin Generation Assay

Thrombin generation in inhibitor plasma (70 BU) was determined using for IXAX-1280.0999.0325, IXAX-1441.0999.0325 and AbE IgG4 bispecific antibodies following purification on Protein A followed by cation exchange chromatography. A thrombin generation assay was used as per Example 13. A thrombin peak was observed for all bispecific antibodies, indicating that both IXAX-1280.0999.0325 and IXAX-1441.0999.0325 can functionally restore haemostasis in plasma containing inhibitory alloantibodies to FVIII, in a similar way to AbE.

A dose response for each bispecific antibody was carried out and the peak thrombin height (Cmax) was determined. FIG. 43. In the concentration range analysed, the Cmax dose responses for IXAX-1280.0999.0325 and IXAX-1441.0999.0325 were greater than the Cmax of AbE, indicating greater thrombin burst, and the Tmax dose responses for IXAX-1280.0999.0325 and IXAX-1441.0999.0325 were lower than the Tmax of AbE, indicating faster thrombin burst. FIG. 44.

REFERENCES

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FIXa Binding Arm VH Domain Polypeptide Sequences

TABLE S-9A Anti-FIXa VH domain sequences and CDRs VH VH nucleo- amino Ab tide acid VH HCDR1 HCDR2 HCDR3 sequence sequence N01 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 92H NO: 11 NO: 12 NO: 13 NO: 14 NO: 15 GFTF IKQD AREG GAGGTGCAGC EVQLVESGGG SSYW GSEK YSSY TGGTGGAGTC LVQPGGSLRL YYYG TGGGGGAGGC SCAASGFTFS MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC IKQDGSEKYY TCCTGTGCAG VDSVKGRFTI CCTCTGGATT SRDNAKNSLY CACCTTTAGT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSYYYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAAGCAAG ATGGAAGTGA GAAATACTAT GTGGACTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGCAGTT ACTACTACTA CGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 12H NO: 11 NO: 2 NO: 3 NO: 16 NO: 17 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG SSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAASGFTFS MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKYY TCCTGTGCAG VDSVKGRFTI CCTCTGGATT SRDNAKNSLY CACCTTTAGT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATACTAT GTGGACTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 05H NO: 18 NO: 2 NO: 3 NO: 19 NO: 20 GFIF INQDG AREG GAGGTGCAGC EVQLVESGGG SSYW SEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCVASGFIFS MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKYY TCCTGTGTAG VDSVKGRFTI CCTCTGGATT SRDNAKNSLY CATCTTTAGT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAT ATAAATCAAG ATGGAAGTGA GAAATACTAT GTGGACTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA NO2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 11H NO: 1 NO: 2 NO: 3 NO: 21 NO: 22 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKNSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 03H NO: 23 NO: 2 NO: 24 NO: 25 NO: 26 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NNYW GSEK YTDS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV CTGGTCCAGC NYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFI TCTCTGGATT ISRDNAKNSV CACCTTTAAT YLQMNSLRAE AACTATTGGA DTAVYYCARE TGAGCTGGGT GYTDSSYYGM CCGCCAGGCT DVWGQGTTVS CCAGGGAAGG VSS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCATCATC TCCAGAGACA ACGCCAAAAA TTCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATACCGATT CGTCCTATTA TGGAATGGAC GTCTGGGGCC AAGGGACCAC GGTCTCCGTC TCCTCA N01 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 28H NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 15H NO: 11 NO: 12 NO: 3 NO: 27 NO: 28 GFTF IKQD AREG GAGGTGCAGC EVQLVESGGG SSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAASGFTFS MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC IKQDGSEKYY TCCTGTGCAG VDSVKGRFTI CCTCTGGATT SRDNAKNSLY CACCTTTAGT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAAGCAAG ATGGAAGTGA GAAATACTAT GTGGACTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCACTGTAT CTGCAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGCAGTT CGTCCTACTA CGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 16H NO: 1 NO: 2 NO: 3 NO: 29 NO: 30 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKNSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 17H NO: 1 NO: 2 NO: 3 NO: 31 NO: 32 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKNSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA CTCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 18H NO: 1 NO: 2 NO: 3 NO: 33 NO: 34 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKKSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA ATCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 19H NO: 1 NO: 2 NO: 3 NO: 35 NO: 36 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKNSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 20H NO: 1 NO: 2 NO: 3 NO: 37 NO: 38 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSL SYYG TGGGGGAGGC RLSCAVSGFT MDV TTTGTCCAGC FNSYWMSW CTGGGGGGTC VRQAPGKGLE CCT WVANINQD GAGACTCTCC GSEKFYVASV TGTGCAGTCT KGRFTISR CTGGATTCAC DNAKKSVYVQ CTTTAATAGC MNSLRAED TATTGGATGA TAVYYCAREG GCT YSSSSYYG GGGTCCGCCA MDVWGQGTTV GGCTCCAGGG TVSS AAGGGGCTGG AGTGGGTGGC CAACATAAAC CAA GATGGAAGTG AGAAATTCTA TGTGGCCTCT GTGAAGGGCC GATTCACCAT CTC CAGAGACAAC GCCAAGAAAT CAGTGTATGT ACAAATGAAC AGCCTGAGAG CCG AGGACACGGC TGTGTATTAC TGTGCGAGAG AGGGGTATAG TAGTTCGTCC TAT TATGGTATGG ACGTCTGGGG CCAAGGGACC ACGGTCACCG TCTCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 21H NO: 1 NO: 2 NO: 3 NO: 39 NO: 40 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKNSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA CTCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 22H NO: 1 NO: 2 NO: 3 NO: 41 NO: 42 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 23H NO: 1 NO: 2 NO: 3 NO: 43 NO: 44 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 24H NO: 1 NO: 2 NO: 3 NO: 45 NO: 46 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKNSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA CTCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 25H NO: 1 NO: 2 NO: 3 NO: 47 NO: 48 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKKSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA ATCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 26H NO: 1 NO: 2 NO: 3 NO: 49 NO: 50 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC FVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKNSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 27H NO: 1 NO: 2 NO: 3 NO: 51 NO: 52 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT LQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT CTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 28H NO: 1 NO: 2 NO: 3 NO: 53 NO: 54 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKNSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA CTCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 29H NO: 1 NO: 2 NO: 3 NO: 55 NO: 56 GFTF INQD AREG GAGGTGCAGC EVQLVESGGG NSYW GSEK YSSS TGGTGGAGTC LVQPGGSLRL SYYG TGGGGGAGGC SCAVSGFTFN MDV TTGGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTI TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA SEQ ID SEQ ID SEQ ID NO: 140 NO: 141 NO: 142 HCDR1 HCDR2 HCDR3 consensus consensus consensus GFTFSSYW INQDGSEK AREGYSSSSYY I NN  K GMDVTDYY GF(T/I)F I(N/K)QDGS AREGY(S/T) (S/N)(S/N) EK (S/D)(S/Y) YW (S/Y)YYGMDV N0420H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: NO: 238 NO: 314 161 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YASS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YASSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATGCCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 21H NO: 1 NO: 2 NO: NO: 239 NO: 315 162 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSAS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSASSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTGCCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 22H NO: 1 NO: 2 NO: NO: 240 NO: 316 163 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSA TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSASYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTG CCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 23H NO: 1 NO: 2 NO: NO: 241 NO: 317 164 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN AYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSAYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGGCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 30H NO: 1 NO: 2 NO: NO: 242 NO: 318 165 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSC TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSCSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT GCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 31H NO: 1 NO: 2 NO: NO: 243 NO: 319 166 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSD TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSDSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTG ACTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 32H NO: 1 NO: 2 NO: NO: 244 NO: 320 167 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSE TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSESYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTG AGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 33H NO: 1 NO: 2 NO: NO: 245 NO: 321 168 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSF TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSFSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT TCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 34H NO: 1 NO: 2 NO: NO: 246 NO: 322 169 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSG TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSGSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTG GCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 35H NO: 1 NO: 2 NO: NO: 247 NO: 323 170 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSH TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSHSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTC ACTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 36H NO: 1 NO: 2 NO: NO: 248 NO: 324 171 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSI TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSISYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 37H NO: 1 NO: 2 NO: NO: 249 NO: 325 172 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSK TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSKSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA AGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 38H NO: 1 NO: 2 NO: NO: 250 NO: 326 173 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSL TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSLSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTC TGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 39H NO: 1 NO: 2 NO: NO: 251 NO: 327 174 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSM TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSMSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 40H NO: 1 NO: 2 NO: NO: 252 NO: 328 175 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSN TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSNSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA ACTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 41H NO: 1 NO: 2 NO: NO: 253 NO: 329 176 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSP TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSPSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTC CCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 42H NO: 1 NO: 2 NO: NO: 254 NO: 340 177 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSQ TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSQSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTC AGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 43H NO: 1 NO: 2 NO: NO: 255 NO: 341 178 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSR TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSRSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA GATCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 44H NO: 1 NO: 2 NO: NO: 256 NO: 342 179 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSST TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSTSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA CCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 45H NO: 1 NO: 2 NO: NO: 257 NO: 343 180 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSV TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSVSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTG TGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 46H NO: 1 NO: 2 NO: NO: 258 NO: 344 181 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSW TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSWSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT GGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 47H NO: 1 NO: 2 NO: NO: 259 NO: 345 182 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSY TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSYSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT ACTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 48H NO: 1 NO: 2 NO: NO: 260 NO: 346 183 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN CYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSCYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTGCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 49H NO: 1 NO: 2 NO: NO: 261 NO: 347 184 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN DYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSDYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGGACTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 50H NO: 1 NO: 2 NO: NO: 262 NO: 348 185 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN EYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSEYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGGAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 51H NO: 1 NO: 2 NO: NO: 263 NO: 349 186 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN FYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSFYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTTCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 52H NO: 1 NO: 2 NO: NO: 264 NO: 350 187 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN GYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSGYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGGGCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 53H NO: 1 NO: 2 NO: NO: 265 NO: 351 188 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN HYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSHYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGCACTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 54H NO: 1 NO: 2 NO: NO: 266 NO: 352 189 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN IYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSIYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGATCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 55H NO: 1 NO: 2 NO: NO: 267 NO: 353 190 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN KYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 56H NO: 1 NO: 2 NO: NO: 268 NO: 354 191 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN LYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSLYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGCTGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 57H NO: 1 NO: 2 NO: NO: 269 NO: 355 192 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN MYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSMYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGATGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 58H NO: 1 NO: 2 NO: NO: 270 NO: 356 193 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN NYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSNYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGAACTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 59H NO: 1 NO: 2 NO: NO: 271 NO: 357 194 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN PYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSPYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGCCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 60H NO: 1 NO: 2 NO: NO: 272 NO: 358 195 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN QYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSQYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGCAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 61H NO: 1 NO: 2 NO: NO: 273 NO: 359 196 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN RYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSRYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGAGATATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 62H NO: 1 NO: 2 NO: NO: 274 NO: 360 197 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN TYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSTYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGACCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 63H NO: 1 NO: 2 NO: NO: 275 NO: 361 198 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN VYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSVYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGGTGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 64H NO: 1 NO: 2 NO: NO: 276 NO: 362 199 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN WYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSWYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTGGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 65H NO: 1 NO: 2 NO: NO: 277 NO: 363 200 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSSS TGGGGGAGGC SCAVSGFTFN YYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSSYYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTT CGTACTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 67H NO: 1 NO: 2 NO: NO: 278 NO: 364 201 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YCSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YCSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATTGCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 68H NO: 1 NO: 2 NO: NO: 279 NO: 365 202 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YDSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YDSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATGACAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 69H NO: 1 NO: 2 NO: NO: 280 NO: 366 203 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YESS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YESSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATGAGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 70H NO: 1 NO: 2 NO: NO: 281 NO: 367 204 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YFSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YFSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATTTCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 71H NO: 1 NO: 2 NO: NO: 282 NO: 368 205 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YGSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YGSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATGGCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 72H NO: 1 NO: 2 NO: NO: 283 NO: 369 206 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YHSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YHSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATCACAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 73H NO: 1 NO: 2 NO: NO: 284 NO: 370 207 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YISS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YISSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATATCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 74H NO: 1 NO: 2 NO: NO: 285 NO: 371 208 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YKSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YKSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAAGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 75H NO: 1 NO: 2 NO: NO: 286 NO: 372 209 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YLSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YLSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATCTGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 76H NO: 1 NO: 2 NO: NO: 287 NO: 373 210 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YMSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YMSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATATGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 77H NO: 1 NO: 2 NO: NO: 288 NO: 374 211 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YNSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YNSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAACAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 78H NO: 1 NO: 2 NO: NO: 289 NO: 375 212 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YPSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YPSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAA TGGAAGTGAG AAATTCTATG TGGCCTCTGT GAAGGGCCGA TTCACCATGT CCAGAGACAA CGCCAAGAAA TCAGTGTATG TACAAATGAA CAGCCTGAGA GCCGAGGACA CGGCTGTGTA TTACTGTGCG AGAGAGGGGT ATCCCAGTTC GTCCTATTAT GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 79H NO: 1 NO: 2 NO: NO: 290 NO: 376 213 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YQSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YQSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATCAGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 80H NO: 1 NO: 2 NO: NO: 291 NO: 377 214 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YRSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YRSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGAAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 81H NO: 1 NO: 2 NO: NO: 292 NO: 378 215 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YTSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YTSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATACCAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 82H NO: 1 NO: 2 NO: NO: 293 NO: 379 216 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YVSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YVSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATGTGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 83H NO: 1 NO: 2 NO: NO: 294 NO: 380 217 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YWSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YWSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATTGGAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 84H NO: 1 NO: 2 NO: NO: 295 NO: 381 218 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YYSS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YYSSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATTACAGTT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 85H NO: 1 NO: 2 NO: NO: 296 NO: 382 219 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSCS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSCSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTTGCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 86H NO: 1 NO: 2 NO: NO: 297 NO: 383 220 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSDS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSDSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTGACT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 87H NO: 1 NO: 2 NO: NO: 298 NO: 384 221 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSES TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSESSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTGAGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 88H NO: 1 NO: 2 NO: NO: 299 NO: 385 223 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSFS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSFSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTTTCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 89H NO: 1 NO: 2 NO: NO: 300 NO: 386 224 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSGS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSGSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTGGCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 90H NO: 1 NO: 2 NO: NO: 301 NO: 387 225 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSHS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSHSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTCACT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 91H NO: 1 NO: 2 NO: NO: 302 NO: 388 226 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSIS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSISSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTATCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 92H NO: 1 NO: 2 NO: NO: 303 NO: 389 227 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSKS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSKSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAAGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 93H NO: 1 NO: 2 NO: NO: 304 NO: 390 228 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSLS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSLSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTCTGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 94H NO: 1 NO: 2 NO: NO: 305 NO: 391 229 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSMS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSMSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTATGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 95H NO: 1 NO: 2 NO: NO: 306 NO: 392 230 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSNS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSNSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAACT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 96H NO: 1 NO: 2 NO: NO: 307 NO: 393 231 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSPS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSPSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTCCCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 97H NO: 1 NO: 2 NO: NO: 308 NO: 394 232 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSQS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSQSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTCAGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 98H NO: 1 NO: 2 NO: NO: 309 NO: 395 233 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSRS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSRSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGAT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N04 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 99H NO: 1 NO: 2 NO: NO: 310 NO: 396 234 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSTS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSTSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTACCT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N05 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 00H NO: 1 NO: 2 NO: NO: 311 NO: 397 235 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSVS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSVSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTGTGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N05 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 01H NO: 1 NO: 2 NO: NO: 312 NO: 398 236 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSWS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSWSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTTGGT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG N05 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 02H NO: 1 NO: 2 NO: NO: 313 NO: 399 237 GAGGTGCAGC EVQLVESGGG AREG TGGTGGAGTC FVQPGGSLRL YSYS TGGGGGAGGC SCAVSGFTFN SYYG TTTGTCCAGC SYWMSWVRQA MDV CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSYSSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTTACT CGTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAG SEQ ID NO: 400 Consensus HCDR3 of N436 and selected variants with parent N128H APEGYSSXSYYGMDV X is I, L, V, R, W, Q, K, H, E, N, M, S Representing the N436H CDR3 sequence AREG YSSISYYGMDV in which the Ile is retained or replaced by Leu, Val, Arg, Trp, Gln, Lys, His, Glu, Asn, Met or Ser. SEQ ID NO: 401 Consensus HCDR3 of N436 and  selected variants AREG YSSXSYYGMDV X is I, L, V, R, W, Q, K, H, E, N or M Representing the N436H CDR3 sequence AREGYSSISYYG MDV in which the Ile is retained or replaced by Leu, Val, Arg, Trp, Gln, Lys, His, Glu, Asn or Met. SEQ ID NO: 402 Consensus HCDR3 of N436 and selected hydrophooic or positively charged variants AREGYSSXSYYGMDV X is I, L, V, R, W, Q or K Representing the N436H CDR3 sequence AREG YSSISYYGMDV in which the Ile is retained or replaced by  Leu, Val, Arg, Trp, Gln or Lys. SEQ ID NO: 403 Consensus HCDR3 of initial most active variants AREG YSSX SYYG MDV X is I, L or V Representing the N436H CDR3 sequence AREGYSSISYYGMDV in which the Ile is retained or replaced by Leu or Val. SEQ ID NO: 406 Consensus HCDR1 GFXFNSYW X is T or R Representing the N1280H CDR1 sequence GFRFNSYW (SEQ ID NO: 441) in which the Arg is retained or replaced by Thr. SEQ ID NO: 407 Consensus HCDR2 INQX₁GX₂X₃K X1 is D, G or W. X2 is S or F. X3 is E or R. Representing the N1280H CDR2 sequence INQDGSRK (SEQ ID NO: 436) in which the Asp is retained or replaced by Gly or Trp, the Ser is retained or replaced by Phe and the Arg is retained or replaced by Glu. SEQ ID NO: 634 Consensus HCDR 2 INQDGSXK X is R or E. Representing the N1280H CDR2 sequence INQDGSRK (SEQ ID NO: 436) in which the Arg is retained or replaced by Glu. SEQ ID NO: 408 Consensus HCDR 3 AREGYSSX₁X₂YYGMDV X1 is S or I. X2 is S or K. Representing the N1280H CDR3 sequence AREGYSSIKYYGMDV (SEQ ID NO: 433) in which the Ile is retained or replaced by Ser and the Lys is retained or replaced by Ser. SEQ ID NO: 635 Consensus HCDR3 AREGYSSIXYYGMDV X is K or S. Representing the N1280H CDR3 sequence AREGYSSIKYYGMDV (SEQ ID NO: 433) in which the Lys is retained or replaced by Ser. N05 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 11H NO: 1 NO: 2 NO: NO: 434 NO: 435 433 GAGGTGCAGC EVQLVESGGG TGGTGGAGTC FVQPGGSLRL TGGGGGAGGC SCAVSGFTFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC IKQDGSEKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSIKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTGA GAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N10 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 91H NO: 1 NO: NO: NO: 437 NO: 438 436 171 GAGGTGCAGC EVQLVESGGG TGGTGGAGTC FVQFGGSLRL TGGGGGAGGC SCAVSGFTFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSRKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSISSYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTAG AAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCTCCTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N11 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 72H NO: 1 NO: NO: NO: 439 NO: 440 436 433 GAGGTGCAGC EVQLVESGGG TGGTGGAGTC FVQPGGSLRL TGGGGGAGGC SCAVSGFTFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSRKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CACCTTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSIKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTAG AAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N12 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 80H NO: NO: NO: NO: 442 NO: 443 441 436 433 GAGGTGCAGC EVQLVESGGG TGGTGGAGTC FVQPGGSLRL TGGGGGAGGC SCAVSGFRFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGSRKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CAGATTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSIKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGAAGTAG AAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N13 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 14H NO: NO: NO: NO: 445 NO: 446 441 444 433 GAGGTGCAGC EVQLVESGGG INQG TGGTGGAGTC FVQPGGSLRL GSRK TGGGGGAGGC SCAVSGFRFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQGGSRKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CAGATTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSIKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG GCGGAAGTAG AAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N13 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 27H NO: 441 NO: 447 NO: 433 NO: 448 NO: 449 INQW GAGGTGCAGC EVQLVESGGG GSRK TGGTGGAGTC FVQPGGSLRL TGGGGGAGGC SCAVSGFRFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQWGSRKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CAGATTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSIKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAT GGGGAAGTAG AAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N13 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 33H NO: 441 NO: 450 NO: 433 NO: 451 NO: 452 INQD GAGGTGCAGC EVQLVESGGG GFRK TGGTGGAGTC FVQPGGSLRL TGGGGGAGGC SCAVSGFRFN TTTGTCCAGC SYWMSWVRQA CTGGGGGGTC PGKGLEWVAN CCTGAGACTC INQDGFRKFY TCCTGTGCAG VASVKGRFTM TCTCTGGATT SRDNAKKSVY CAGATTTAAT VQMNSLRAED AGCTATTGGA TAVYYCAREG TGAGCTGGGT YSSIKYYGMD CCGCCAGGCT VWGQGTTVTV CCAGGGAAGG SS GGCTGGAGTG GGTGGCCAAC ATAAACCAAG ATGGATTCAG AAAATTCTAT GTGGCCTCTG TGAAGGGCCG ATTCACCATG TCCAGAGACA ACGCCAAGAA ATCAGTGTAT GTACAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT ATTACTGTGC GAGAGAGGGG TATAGTAGTA TCAAGTATTA TGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCA N14 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 54H NO: 441 NO: 436 NO: 433 NO: 453 NO: 454 GAGGTGCAGC EVQLVESGGG TGGTTGAATC FVQPGGSLRL TGGCGGCGGA SCAVSGFRFN TTTGTTCAGC SYWMSWVRQA CTGGCGGCTC PGKGLEWVAN TCTGAGACTG INQDGSRKFY AGCTGTGCCG VASVKGRFTM TGTCCGGCTT SRDNAKKEVY CCGGTTCAAC VQMNSLRAED AGCTACTGGA TAVYYCAREG TGTCCTGGGT YSSIKYYGMD CCGACAGGCC VWGQGTTVTV CCTGGCAAAG SS GACTTGAGTG GGTCGCCAAC ATCAACCAGG ACGGCAGCCG GAAGTTTTAC GTGGCCTCTG TGAAGGGCAG ATTCACCATG AGCCGGGACA ACGCCAAGAA AGAGGTGTAC GTGCAGATGA ACAGCCTGAG AGCCGAGGAC ACCGCCGTGT ACTATTGTGC CAGAGAGGGC TACAGCAGCA TCAAGTACTA CGGCATGGAC GTGTGGGGCC AGGGCACAAC AGTGACAGTC TCTTCT N14 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 41H NO: 441 NO:436 NO: 433 NO: 455 NO: 456 GAGGTGCAGC EVQLVESGGG TGGTTGAATC FVQPGGSLRL TGGCGGCGGA SCAVSGFRFN TTTGTTCAGC SYWMSWVRQA CTGGCGGCTC PGKGLEWVAN TCTGAGACTG INQDGSRKFY AGCTGTGCCG VASVKGRFTM TGTCCGGCTT SRDNADKSVY CCGGTTCAAC VQMNSLRAED AGCTACTGGA TAVYYCAREG TGTCCTGGGT YSSIKYYGMD CCGACAGGCC VWGQGTTVTV CCTGGCAAAG SS GACTTGAGTG GGTCGCCAAC ATCAACCAGG ACGGCAGCCG GAAGTTTTAC GTGGCCTCTG TGAAGGGCAG ATTCACCATG AGCCGGGACA ACGCCGACAA AAGCGTGTAC GTGCAGATGA ACAGCCTGAG AGCCGAGGAC ACCGCCGTGT ACTATTGTGC CAGAGAGGGC TACAGCAGCA TCAAGTACTA CGGCATGGAC GTGTGGGGCC AGGGCACAAC AGTGACAGTC TCTTCT N14 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 42H NO: 441 NO: 436 NO: 433 NO: 457 NO: 458 GAGGTGCAGC EVQLVESGGG TGGTTGAATC FVQPGGSLRL TGGCGGCGGA SCAVSGFRFN TTTGTTCAGC SYWMSWVRQA CTGGCGGCTC PGKGLEWVAN TCTGAGACTG INQDGSRKFY AGCTGTGCCG VASVKGRFTM TGTCCGGCTT SRDNAEKSVY CCGGTTCAAC VQMNSLRAED AGCTACTGGA TAVYYCAREG TGTCCTGGGT YSSIKYYGMD CCGACAGGCC VWGQGTTVTV CCTGGCAAAG SS GACTTGAGTG GGTCGCCAAC ATCAACCAGG ACGGCAGCCG GAAGTTTTAC GTGGCCTCTG TGAAGGGCAG ATTCACCATG AGCCGGGACA ACGCCGAGAA AAGCGTGTAC GTGCAGATGA ACAGCCTGAG AGCCGAGGAC ACCGCCGTGT ACTATTGTGC CAGAGAGGGC TACAGCAGCA TCAAGTACTA CGGCATGGAC GTGTGGGGCC AGGGCACAAC AGTGACAGTC TCTTCT

TABLE S-9B Anti-FIXa VH domain framework sequences Ab VH FR1 FR2 FR3 FR4 N0192H SEQ ID SEQ ID SEQ ID SEQ ID NO: 148 NO: 133 NO: 149 NO: 135 EVQL YYVD VESG SVKG GGLV RFTI QPGG SRDN SLRL AKNS SCAA LYLQ S MNSL RAED TAVY YC N0212H SEQ ID SEQ ID SEQ ID SEQ ID NO: 148 NO: 133 NO: 149 NO: 135 N0205H SEQ ID SEQ ID SEQ ID SEQ ID NO: 150 NO: 133 NO: 149 NO: 135 EVQL VESG GGLV QPGG SLRL SCVA S N0211H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 152 NO: 135 EVQL FYVA VESG SVKG GGLV RFTI QPGG SRDN SLRL AKNS SCAV VYLQ S MNSL RAED TAVY YC N0203H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 153 NO: 154 FYVA WGQG SVKG TTVS RFII VSS SRDN AKNS VYLQ MNSL RAED TAVY YC N0128H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 EVQL MSWV FYVA WGQG VESG RQAP SVKG TTVT GGFV GKGL RFTM VSS QPGG EWVA SRDN SLRL N AKKS SCAV VYVQ S MNSL RAED TAVY YC N0215H SEQ ID SEQ ID SEQ ID SEQ ID NO: 148 NO: 133 NO: 149 NO: 135 N0216H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 152 NO: 135 N0217H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 155 NO: 135 FYVA SVKG RFTM SRDN AKNS VYLQ MNSL RAED TAVY YC N0218H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 156 NO: 135 FYVA SVKG RFTI SRDN AKKS VYLQ MNSL RAED TAVY YC N0219H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 157 NO: 135 FYVA SVKG RFTI SRDN AKNS VYVQ MNSL RAED TAVY YC N0220H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 158 NO: 135 FYVA SVKG RFTI SRDN AKKS VYVQ MNSL RAED TAVY YC N0221H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 159 NO: 135 FYVA SVKG RFTM SRDN AKNS VYVQ MNSL RAED TAVY YC N0222H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 160 NO: 135 FYVA SVKG RFTM SRDN AKKS VYLQ MNSL RAED TAVY YC N0223H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 134 NO: 135 N0224H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 155 NO: 135 N0225H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 156 NO: 135 N0226H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 157 NO: 135 N0227H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 160 NO: 135 N0228H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 159 NO: 135 N0229H SEQ ID SEQ ID SEQ ID SEQ ID NO: 151 NO: 133 NO: 158 NO: 135 N0511H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1091H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1172H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1280H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1314H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1327H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1333H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 134 NO: 135 N1441H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 459 NO: 135 FYVA SVKG RFTM SRDN ADKS VYVQ MNSL RAED TAVY YC N1442H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 460 NO: 135 FYVA SVKG RFTM SRDN AEKS VYVQ MNSL RAED TAVY YC N1454H SEQ ID SEQ ID SEQ ID SEQ ID NO: 132 NO: 133 NO: 461 NO: 136 FYVA SVKG RFTM SRDN AKKE VYVQ MNSL RAED TAVY YC

FX Binding Arm VH Domain Polypeptide Sequences

TABLE S-10A Anti-FX VH domain sequences and CDRs VH VH  nucleo- amino tide acid se- se- Ab VH HCDR1 HCDR2 HCDR3 quence quence T02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 57 NO: 58 NO: 59 NO: 60 NO: 61 GYTF INAG ARDWAA CAGG QVQL TNYA NGFT AISYYG TCCA VQSG MDV GCTT AEVK GTGC RPGA AGTC SVKV TGGG SCKA GCTG SGYT AGGT FTNY GAAG AIHW AGGC VRQA CTGG PGQR GGCC LEWM TCAG GWIN TGAA AGNG GGTT FTKS TCCT SQKF GCAA RGRV GGCT TITR TCTG DTSA GATA NTAY CACC MELS TTCA SLRS CTAA EDTA CTAT IYYC GCTA ARDW TACA AAAI TTGG SYYG GTGC MDVW GCCA GQGT GGCC TVTV CCCG SS GACA GAGG CTTG AGTG GATG GGAT GGAT CAAC GCTG GCAA TGGT TTCA CAAA ATCT TCAC AGAA GTTC CGGG GCAG AGTC ACCA TTAC CAGG GACA CATC CGCG AACA CAGC CTAC ATGG AACT GAGC AGCC TCAG ATCT GAAG ACAC GGCT ATTT ATTA CTGT GCGA GAGA TTGG GCTG CTGC TATC TCTT ACTA CGGT ATGG ACGT CTGG GGCC AAGG GACC ACGG TCAC CGTC TCCT CAG T05 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 67 NO: 68 NO: 69 NO: 70 NO: 71 GFTF IWYD ARSGYS CAGG QVQL SSYG GTNK SSWYGA TGCA VESG MDV GCTG GGVV GTGG QPGR AGTC SLRL TGGG SCAA GGAG SGFT GCGT FSSY GGTC GMHW CAGC VRQA CTGG PGEG GAGG LEWV TCCC AVIW TGAG YDGT ACTC NKYY TCCT ADSL GTGC KGRF AGCG TISR TCTG DNSK GATT NTLY CACC LQMN TTCA RLRA GTAG EDTA CTAT VYYC GGCA ARSG TGCA YSSS CTGG WYGA GTCC MDVW GCCA GQGT GGCT TVTV CCAG SS GCGA GGGG CTGG AGTG GGTG GCAG TTAT ATGG TATG ATGG AACT AATA AATA CTAT GCAG ACTC CTTG AAGG GCCG ATTC ACCA TCTC CAGA GACA ATTC CAAG AACA CGCT CTAT CTGC AAAT GAAC AGGC TGAG AGCC GAGG ACAC GGCT GTGT ATTA CTGT GCGA GGTC CGGG TATA GCAG CAGC TGGT ACGG CGCT ATGG ACGT CTGG GGCC AAGG GACC ACGG TCAC CGTC TCCT CAG T06 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 77 NO: 78 NO: 79 NO: 80 NO: 81 GYTF INAG ARDWAA CAGG QVQL TSYA NGIT AITYYG TCCA VQSG MDV GCTT AEVK GTGC RPGA AGTC SVKV TGGG SCKA GCTG SGYT AGGT FTSY GAAG AIHW AGGC VRQA CTGG PGQR GGCC LEWM TCAG GWIN TGAA AGNG GGTT ITKS TCCT SQKF GCAA QGRV GGCT TITR TCTG DTSA GATA NTVY CACC LELS TTCA SLRS CAAG EDTA CTAC VYYC GCCA ARDW TACA AAAI TTGG TYYG GTGC MDVW GCCA GQGT GGCC TVTV CCCG SS GACA GAGG CTTG AGTG GATG GGAT GGAT CAAC GCTG GCAA TGGT ATCA CAAA ATCT TCAC AGAA GTTC CAGG GCAG AGTC ACCA TTAC CAGG GACA CATC CGCG AACA CAGT TTAC CTGG AACT GAGC AGCC TCAG ATCT GAAG ACAC GGCT GTTT ATTA TTGT GCGA GAGA TTGG GCTG CTGC TATC ACCT ACTA CGGT ATGG ACGT CTGG GGCC AAGG GACC ACGG TCAC CGTC TCCT CAG T12 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 86 NO: 87 NO: 88 NO: 89 NO: 90 EFTF ISYDG AKDFTM CAGG QVQL STAG SNK VRGVII TGCA VESG MDV GCTG GGVL GTGG QPGK AGTC SLRL TGGG SCAA GGGG SEFT GCGT FSTA ACTC GMHW CAGC VRQA CTGG PGKG GAAG LEWV TCCC TFIS TGAG YDGS ACTC NKYY TCCT ADSV GTGC KGRF AGCC TISR TCTG DNSK AATT VYLQ CACC MNSL TTCA RTED GTAC TAVY CGCT YCAK GGCA DFTM TGCA VRGV CTGG IIMD GTCC VWGQ GCCA GTTV GGCT TVSS CCAG GCAA GGGG CTGG AGTG GGTG ACTT TTAT ATCA TATG ATGG AAGT AATA AATA CTAT GCAG ACTC CGTG AAGG GCCG ATTC ACCA TCTC CAGA GACA ATTC CAAG GTGT ATCT GCAA ATGA ACAG CCTG AGAA CTGA GGAC ACGG CTGT GTAT TACT GTGC GAAA GATT TCAC TATG GTTC GGGG AGTT ATTA TAAT GGAC GTCT GGGG CCAA GGGA CCAC GGTC ACCG TCTC CTCA G T14 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 96 NO: 97 NO: 98 NO: 99 NO: 100 GGSI IYYS AKGA CAGG QVQL SSYY GST AGDY TGCA QESG GCTG PGLV CAGG KPSE AGTC TLSL GGGC TCTV CCAG SGGS GACT ISSY GGTG YWSW AAGC IRQP CTTC PGKG GGAG LEWI ACCC GYIY TGTC YSGS CCTC TNYN ACCT PSLK GCAC SRVN TGTC ISVD TCTG TSKN GTGG QFSL CTCC RLSS ATCA VTAA GTAG DTAV TTAT YYCA TACT KGAA GGAG GDYW CTGG GQGT ATCC LVTV GGCA SS GCCC CCAG GGAA GGGA CTGG AGTG GATT GGGT ATAT CTAT TACA GTGG GAGC ACCA ACTA TAAC CCCT CCCT CAAG AGTC GAGT CAAC ATAT CAGT AGAC ACGT CCAA GAAC CAGT TCTC CCTG AGGC TGAG TTCT GTGA CCGC TGCG GACA CGGC CGTG TATT ATTG TGCG AAAG GGGC AGCT GGGG ACTA CTGG GGCC AGGG AACC CTGG TCAC CGTC TCCT CAG T15 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 105 NO: 106 NO: 107 NO: 108 NO: 109 GGSI IYYS ARGL CAGG QVQL SKYY GNT GDY TGCA QESG GCTG PGLV CAGG KPSE AGTC TLSL GGGC TCTV CCAG SGGS GACT ISKY GGTG YWSW AAGC IRQP CTTC PGKG GGAG LEWI ACCC GYIY TGTC YSGN CCTC TYQN ACCT PSLK GCAC SRVT TGTC ISID TCTG GTGG TSKN CTCC QISL ATCA KVSS GTAA VTAA ATAC DTAV TACT YYCA GGAG RGLG CTGG DYWG ATCC QGTL GGCA VTVS GCCC S CCAG GGAA GGGA CTGG AGTG GATT GGAT ATAT CTAT TACA GTGG GAAC ACCT ACCA GAAT CCCT CCCT CAAG AGTC GAGT CACC ATAT CAAT AGAC ACGT CCAA GAAC CAGA TCTC CCTG AAGG TGAG CTCT GTGA CCGC TGCG GACA CGGC CGTC TATT ACTG TGCG AGAG GGCT GGGG GACT ACTG GGGC CAGG GAAC CCTG GTCA CCGT CTCC TCAG T23 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 114 NO: 115 NO: 116 NO: 117 NO: 118 GGSI IYYS ARGL CAGG QVQL SRYY GTT GDF TGCA QESG GCTG PGLV CAGG KPSE AGTC TLSL GGGC TCSV CCAG SGGS GACT ISRY GGTG YWSW AAGC IRQP CTTC PGKG GGAG LEWI ACCC GYIY TGTC YSGT CCTC TYYN ACCT PSLK GCAG SRVT TGTC FSVD TCTG TSKT GTGG QFSL CTCC KLNS ATTA VTAA GTAG DTAV ATAT YYCA TACT RGLG GGAG DFWG CTGG RGTL ATCC VTVS GGCA S GCCC CCAG GGAA GGGA CTGG AGTG GATT GGAT ATAT CTAT TACA GTGG GACC ACCT ACTA TAAC CCCT CCCT CAAG AGTC GAGT CACC TTTT CAGT AGAC ACGT CCAA GACC CAGT TCTC CCTG AAAC TTAA CTCT GTGA CCGC TGCG GACA CGGC CGTA TATT ACTG TGCG AGAG GACT GGGG GACT TCTG GGGC CGGG GAAC CCTG GTCA CCGT CTCC TCAG T25 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 122 NO: 123 NO: 124 NO: 125 NO: 126 GGSIS INNS ARGG CAGG QVQL SGIYY GNT SGDY TGCA QESG GCTG PGLV CAGG KPSE AGTC TLSL GGGC TCTV CCAG SGGS GACT ISSG GGTG IYYW AAGC SWIR CTTC QHPG AGAG KGLE ACCC WIGY TGTC INNS CCTC GNTY ACCT YNPS GCAC LKGR TGTC VNIS TCTG VDTS GTGG KKQF CTCC SLKL ATCA SSVT GTAG DADT TGGT AVYY ATAT CARG ACTA GSGD CTGG YWGQ AGTT GTLV GGAT TVSS CCGC CAGC ACCC AGGG AAGG GCCT GGAG TGGA TTGG ATAC ATCA ATAA CAGT GGGA ACAC CTAC TACA ACCC GTCC CTCA AGGG TCGA GTTA ACAT ATCA GTAG ACAC GTCT AAGA AACA GTTC TCCC TGAA GCTG AGCT CTGT GACT GACG CGGA CACG GCCG TCTA TTAC TGTG CGAG GGGG GGAT CGGG CGAC TACT GGGG CCAG GGAA CCCT GGTC ACCG TCTC CTCA G

TABLE S-10B Anti-FX VL domain sequences and CDRs VL  nucleo- amino  tide  acid se- se- Ab VL LCDR1 LCDR2 LCDR3 quence quence T02 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 62 NO: 63 NO: 64 NO: 65 NO: 66 SSNI RNT ATWDD CAGTC QSVLT GSNY SLSAYV TGTCC QPPSA TGACT SGTPG CAGCC QRVTI ACCCT SCSGS CAGCG SSNIG TCTGG SNYVY GACCC WYQQL CCGGG PGTAP CAGAG KLLIY GGTCA RNTQR CCATC PSEVP TCTTG DRFSG TTCTG SKSGA GAAGC SASLA AGCTC ISGLR CAACA SEDET TCGGA DYYCA AGTAA TWDDS TTATG LSAYV TATAC FGTGT TGGTA KVTVL CCAGC AGCTC CCAGG AACGG CCCCC AAACT CCTCA TCTAT AGGAA TACTC AGCGG CCCTC AGAGG TCCCT GACCG ATTCT CTGGC TCCAA GTCTG GCGCC TCAGC CTCCC TGGCC ATCAG TGGGC TCCGG TCCGA GGATG AGACT GATTA TTACT GTGCA ACATG GGATG ACAGC CTGAG TGCTT ATGTC TTCGG AACTG GGACC AAAGT CACCG TCCTA G T05 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 72 NO: 73 NO: 74 NO: 75 NO: 76 SSDVG EVN SSYAG CAGTC QSALT GYYY SNTWV TGCCC QPPSA TGACT SGSPG CAGCC QSVTI TCCCT SCTGT CCGCG SSDVG TCCGG GYYYV GTCTC SWYQQ CTGGA HPGKA CAGTC PKLMI AGTCA YEVNK CCATC RPSGV TCCTG PDRFS CACTG GSKSG GAACC ITASL AGCAG TVSGL TGACG QSEDE TTGGT ADYYC GGTTA SSYAG TTACT SNTWV ATGTC FGGGT TCCTG KLTVL GTACC AACAG CACCC AGGCA AAGCC CCCAA ACTCA TGATT TATGA GGTCA ATAAG CGGCC CTCAG GGGTC CCTGA TCGCT TCTCT GGCTC CAAGT CTGGC ATCAC GGCCT CCCTG ACCGT CTCTG GGCTC CAGTC TGAGG ATGAG GCTGA TTATT ACTGC AGCTC ATATG CAGGC AGCAA CACTT GGGTG TTCGG CGGAG GGACC AAGCT GACCG TCCTA G T06 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 62 NO: 82 NO: 83 NO: 84 NO: 85 SSNI RNN FGAGT CAGTC QSVLT GSNY KVTVL TGTGC QPPSV TGACT SGTPG CAGCC QRVTI ACCCT SCSGS CAGTG SSNIG TCTGG SNYVY GACCC WYQQF CCGGG PGTAP CAGAG KLLIY GGTCA RNNQR CCATC PSEVP TCTTG DRFSG TTCTG SKSGA GAAGC SASLA AGCTC ISGLR CAACA SEDET TCGGA DYYCA AGTAA TWDDS TTATG LSAYV TATAC FGAGT TGGTA KVTVL CCAGC AGTTC CCAGG AACGG CCCCC AAACT CCTCA TCTAT AGGAA TAATC AGCGG CCCTC AGAGG TCCCT GACCG ATTCT CTGGC TCCAA GTCTG GCGCC TCAGC CTCCC TGGCC ATCAG TGGGC TCCGG TCCGA GGATG AGACT GATTA TTACT GTGCA ACATG GGATG ACAGC CTGAG TGCTT ATGTC TTCGG AGCTG GGACC AAAGT CACCG TCCTA G T12 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 91 NO: 92 NO: 93 NO: 94 NO: 95 QDISNY DAS QQYDN GACAT DIQMT LPIT CCAGA QSPSS TGACC LSVSV CAGTC GDRVT TCCAT ITCQA CCTCC SQDIS CTGTC NYLNW TGTAT YQQKP CTGTA GKAPK GGAGA LLIYD CAGAG ASNLE TCACC TGVPS ATCAC RFSGS TTGCC GSGTD AGGCG FTFII AGTCA SSLQP GGACA EDIAT TTAGC YYCQQ AACTA YDNLP TTTAA ITFGQ ATTGG GTRLE TATCA IK GCAGA AACCA GGGAA AGCCC CTAAG CTCCT GATCT ACGAT GCATC CAATT TGGAA ACAGG GGTCC CATCA AGGTT CAGTG GAAGT GGATC TGGGA CAGAT TTTAC TTTCA TCATC AGCAG CCTGC AGCCT GAAGA TATTG CAACA TATTA CTGTC AACAG TATGA TAATC TCCCG ATCAC CTTCG GCCAA GGGAC ACGAC TGGAG ATCAA AC T14 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 101 NO: 92 NO: 102 NO: 103 NO: 104 QSVNSY DAS QQRNN GAAAT EIVLA WPIT TGTGT QSPAT TGGCA LSLSP CAGTC GERAT TCCAG FSCRA CCACC SQSVN CTGTC SYLAW TTTGT HQQKP CTCCA GQAPR GGGGA LLIYD AAGAG ASNRA CCACG TGIPA TTCTC RFSGS CTGCA GSGTD GGGCC FTLTI AGTCA SSLEP GAGTG EDFAV TTAAC YYCQQ AGCTA RNNWP CTTAG ITFGQ CCTGG GTRLE CACCA IK ACAGA AACCT GGCCA GGCTC CCAGG CTCCT CATCT ATGAT GCATC CAACA GGGCC ACTGG CATCC CAGCC AGGTT CAGTG GCAGT GGGTC CGGGA CAGAC TTCAC TCTCA CCATC AGCAG CCTAG AGCCT GAAGA TTTTG CAGTT TATTA CTGTC AGCAG CGTAA CAACT GGCCT ATCAC CTTCG GCCAA GGGAC ACGAC TGGAG ATCAA AC T15 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 110 NO: 92 NO: 111 NO: 112 NO: 113 QSVSSY DAS QQRSN GAAAT EIVLT WPLT TGTGT QSPAT TGACA LSLSP CAGTC GERAT TCCAG LSCRA CCACC SQSVS CTGTC SYLAW TTTGT HQQKP CTCCA GQAPR GGGGA LLIYD AAGAG ASNRA CCACC TGIPA CTCTC RFSGS CTGCA GSGTD GGGCC FTLTI AGTCA SSLEP GAGTG EDFAV TTAGC YYCQQ AGCTA RSNWP CTTAG LTFGG CCTGG GTKVE CACCA IK ACAGA AACCT GGCCA GGCTC CCAGG CTCCT CATCT ATGAT GCATC CAACA GGGCC ACTGG CATCC CAGCC AGGTT CAGTG GCAGT GGGTC TGGGA CAGAC TTCAC TCTCA CCATC AGCAG CCTAG AGCCT GAAGA TTTTG CAGTT TATTA CTGTC AGCAA CGTAG CAACT GGCCT CTCAC TTTCG GCGGA GGGAC CAAGG TGGAG ATCAA AC T23 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 119 NO: 92 NO: 111 NO: 120 NO: 121 QSVSGY DAS QQRSN GAAAT EIVLT WPLT TGTGT QSPAT TGACT LSLSP CAGTC GERAT TCCAG LSCRA CCACC SQSVS CTGTC GYLAW ATTGT HQQKP CTCCA GQAPR GGGGA LLIYD AAGGG ASNRA CCACC TGIPA CTCTC RFSGS CTGCC GSGTD GGGCC FTLTI AGTCA SSLEP GAGTG EDFAV TTAGC YYCQQ GGCTA RSNWP CTTAG LTFGG CCTGG GTKVE CACCA IK ACAGA AACCT GGCCA GGCTC CCAGG CTCCT CATCT ATGAT GCATC CAACA GGGCC ACTGG CATCC CAGCC AGATT CAGTG GCAGT GGGTC TGGGA CAGAC TTCAC TCTCA CCATC AGCAG CCTAG AGCCT GAAGA TTTTG CAGTT TATTA CTGTC AGCAA CGTAG CAACT GGCCT CTCAC TTTCG GCGGA GGGAC CAAGG TGGAG ATCAA AC T25 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 128 NO: 92 NO: 129 NO: 130 NO: 131 QSINNY DAS QQRNN GAAAT EIVLT WPPT TGTGT QSPAT TGACA LSLSP CAGTC GERAT TCCAG LSCRT CCACC SQSIN CTGTC NYLAW TTTGT FQQKP CTCCA GQAPR GGGGA LLIYD AAGAG ASNRA CCACC PGIPA CTCTC RFSGS CTGCA GSGTD GGACC FTLTI AGTCA SSLEP GAGTA EDFVV TTAAC YFCQQ AACTA RNNWP CTTAG PTFGQ CCTGG GTKVE TTCCA IK ACAGA AACCT GGCCA GGCTC CCAGG CTCCT CATCT ATGAT GCATC CAACA GGGCC CCTGG CATCC CAGCC AGGTT CAGTG GCAGT GGGTC TGGGA CAGAC TTCAC TCTCA CCATC AGCAG CCTGG AGCCT GAAGA TTTTG TAGTT TATTT CTGTC AGCAG CGTAA CAACT GGCCT CCGAC ATTCG GCCAA GGGAC CAAGG TGGAA ATCAA AC

TABLE S-10C Anti-FX VH domain sequences and CDRs The following TxxxxH VH domains are suitable for pairing with a common light chain VL such as VL domain 0128L or 0325L. VH VH nucleo- Amino tide acid se- se- Ab VH HCDR1 HCDR2 HCDR3 quence quence T0200H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 464 NO: 465 NO: 466 RYSFT 463 ARDGY CAGGT QVQLV SYY INPKT GSSAR GCAGC QSGAE GDT CLQL TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYLH AAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKT AGGTT GDTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TTTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATC CARDG TGCAT YGSSA TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAACT GGTGA CACAA GCTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAC CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGGCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0201H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: 467 NO: 468 NO: 469 NO: 470 INPKS ARDGY CAGGT QVQLI GST GSSSR GCAGT QSGAE CLQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0202H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 471 NO: 472 467 CAGGT QVQLI GCAGC QSGAE TGATA VQKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGCA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0203H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: 467 NO: 468 NO: 473 NO: 474 CAGGT QVQLV GCAGC QSGAE TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELIS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA TCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0204H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: 467 NO: 468 NO: 475 NO: 476 CAGGT QVQLV GCAGC QSGAE TGGTG VQKTG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGCA SYYMH GAAGA WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0205H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 464 NO: 478 NO: 479 477 CAGGT QVQLV INPK GCAGC QSGAE SGDT TGGTG VKKPG CAGTC ASVKV TGGGG S CTGAG CKASR GTGAA YSFTS GAAGC YYMHW CTGGG VRQAP GCCTC G AGTGA QGLEW AGGTT MGIIN TCCTG PKSGD CAAGG TSYAQ CATCT K AGATA FQGRV CAGCT TMTRD TCACC TSTST AGCTA VYMEL CTATA N TGCAC SLRSE TGGGT DTAVY GCGAC YCARD AGGCC GYGSS CCTGG A ACAAG RCLQL GGCTT WGQGT GAGTG LVTVS GATGG S GAATA ATCAA CCCTA AAAGT GGTGA CACAA GCTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA ACAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGGCC CGGTG CCTCC AGCTC TGGGG CCAGG GCGCC CTGGT CACCG TCTCC TCA T0206H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: 477 NO: 464 NO: 480 NO: 481 CAGGT QVQLV GCAGC QSGAE TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GDTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MDLSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSA TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTGA CACAA GCTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA CCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGGCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0207H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 482 NO: 483 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKTG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGA WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0208H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 484 NO: 485 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG EQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GAACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0209H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 464 NO: 486 NO: 487 463 CAGGT QVQLV GCAGC QSGAE TGGTG VQKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGCA SYYLH AAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKT AGGTT GDTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TTTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATC CARDG TGCAT YGSSA TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAACT GGTGA CACAA GCTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAC CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGGCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0210H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 488 NO: 499 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELNS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA ACAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0211H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 500 NO: 501 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MDLSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA CCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0212H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 502 NO: 503 NO: 504 NO: 505 NO: 506 GFSFT INPRS ARDGY CAGGT QVQLV SYY GST GSSSR GCAGC QSGAE CFQY TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG GFSFT GTGAA SYYIH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPRS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS GGATT TNTVY CTCCT MDLSS TCACC LRSED AGCTA TAVYY CTATA CARDG TACAC YGSSS TGGGT RCFQY GCGCC WGQGT AGGCC LVTVS CCTGG S ACAAG GACTT GAGTG GATGG GAATA ATCAA CCCTA GAAGT GGTAG CACAA GCTAC GCTCA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAA CACAG TCTAC ATGGA CCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTAT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGATG CTTCC AGTAC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0213H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 508 NO: 509 507 CAGGT QVQLV INPKS GCAGC QSGAE GTT TGGTG VKKTG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGA WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GTTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAC TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA ACTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0214H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 510 NO: 511 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKTG CAGTC ASVKV TGGGG SCQAS CTGAG RYSFT GTGAA SYYMH GAAGA WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CCAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0215H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 512 NO: 513 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCGGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0216H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 514 NO: 515 467 CAGGT QVQLV GCAGC QSGAE TGGTG VKKTG CAGTC ASVKV TGGGG SCQAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATT CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0217H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 468 NO: 516 NO: 517 467 CAGGT QVQLV GCAGC QSGAE TGGTG VTKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAC SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RCLQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGTG CCTCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0666H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 520 NO: 521 NO: 522 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE IIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RIIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGAT CATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0667H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 523 NO: 524 NO: 525 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE LIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0668H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 526 NO: 527 NO: 528 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE QIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RQIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGCA GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0669H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 529 NO: 530 NO: 531 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE ILML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RILML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGAT CCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0670H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 532 NO: 533 NO: 534 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE LLML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RLLML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGCT GCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0671H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 535 NO: 536 NO: 537 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE QLML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RQLML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGCA GCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0672H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 538 NO: 539 NO: 540 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE IIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RIIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGAT CATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0673H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 541 NO: 542 NO: 543 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE LIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RLIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGCT GATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0674H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 544 NO: 545 NO: 546 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE QIML TGATA VKKPG CAGTC ASVKV TGGGG S CTGAG CKASR GTGAA YSFTS GAAGC YYMHW CTGGG VRQAP GCCTC G AGTGA QGLEW AGGTT MGIIN TCCTG PKSGS CAAGG TSYAQ CATCT K AGATA FQGRV CAGCT TMTRD TCACC TSTST AGCTA VYMEL CTATA S TGCAC SLRSE TGGGT DTAVY GCGAC YCARD AGGCC GYGSS CCTGG S ACAAG RQIML GGCTT WGQGT GAGTG LVTVS GATGG S GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGCA GATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0675H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 547 NO: 548 NO: 549 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE VIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RVIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGGT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0676H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 550 NO: 551 NO: 552 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE VLML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RVLML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGGT GCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0677H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 553 NO: 554 NO: 555 467 ARDGY CAGGT QVQLI GSSSR GCAGT QSGAE VIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSSS TGGGT RVIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT CGTCC CGGGT GATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0678H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 556 NO: 557 NO: 558 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE IIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RIIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGAT CATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0679H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 559 NO: 560 NO: 561 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE ILML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RILML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGAT CCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0680H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 562 NO: 563 NO: 564 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE IIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RIIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGAT CATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0681H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 566 NO: 567 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE LIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0682H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 568 NO: 569 NO: 570 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE LLML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLLML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0683H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: 571 NO: 572 NO: 573 NO: 462 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE LIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0684H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 574 NO: 575 NO: 576 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE QIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RQIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCA GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0685H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 577 NO: 578 NO: 579 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE QLML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RQLML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCA GCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0686H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 580 NO: 581 NO: 582 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE QIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RQIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCA GATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0687H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 583 NO: 584 NO: 585 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE VIQL TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RVIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGGT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0688H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 586 NO: 587 NO: 588 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE VLML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RVLML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGGT GCTCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0689H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 589 NO: 590 NO: 591 467 ARDGY CAGGT QVQLI GSFSR GCAGT QSGAE VIML TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RVIML GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGGT GATCA TGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0713H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 592 NO: NO: 565 NO: 593 NO: 594 RFSFT 467 CAGGT QVQLI SYY GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RFSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATT TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0734H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 595 NO: NO: 565 NO: 596 NO: 597 RYHFT 467 CAGGT QVQLI SYY GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYHFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CCACT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0736H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 598 NO: NO: 565 NO: 599 NO: 600 RYKFT 467 CAGGT QVQLI SYY GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYKFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAAGT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0742H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 601 NO: NO: 565 NO: 602 NO: 603 RYRFT 467 CAGGT QVQLI SYY GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYRFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGAT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0774H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 604 NO: NO: 565 NO: 605 NO: 606 RYSFK 467 CAGGT QVQLI SYY GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFK GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCAAG LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0785H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 607 NO: NO: 565 NO: 608 NO: 609 RYSFT 467 CAGGT QVQLI AYY GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA AYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED GCCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0850H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 611 NO: 612 610 CAGGT QVQLI LNPKS GCAGT QSGAE GST TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA LNPKS AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA CTGAA CCCTA AAAGT GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0925H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 614 NO: 615 613 CAGGT QVQLI INPKI GCAGT QSGAE GST TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKI AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAATC GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0926H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 617 NO: 618 616 CAGGT QVQLI INPKK GCAGT QSGAE GST TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKK AGGTT GSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAAG GGTAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0951H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 620 NO: 621 619 CAGGT QVQLI INPKS GCAGT QSGAE SST TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT SSTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT AGCAG TACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0958H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 623 NO: 624 INPKS 622 CAGGT QVQLI GDT GCAGT QSGAE TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GDTSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTGA CACAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0989H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 626 NO: 627 625 CAGGT QVQLI INPKS GCAGT QSGAE GSR TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSRSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TAGAA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0990H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 462 NO: NO: 565 NO: 629 NO: 630 628 CAGGT QVQLI INPKS GCAGT QSGAE GSS TGATA VKKPG CAGTC ASVKV TGGGG SCKAS CTGAG RYSFT GTGAA SYYMH GAAGC WVRQA CTGGG PGQGL GCCTC EWMGI AGTGA INPKS AGGTT GSSSY TCCTG AQKFQ CAAGG GRVTM CATCT TRDTS AGATA TSTVY CAGCT MELSS TCACC LRSED AGCTA TAVYY CTATA CARDG TGCAC YGSFS TGGGT RLIQL GCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GGCTT GAGTG GATGG GAATA ATCAA CCCTA AAAGT GGTAG TAGCA GTTAC GCACA GAAGT TCCAG GGCAG AGTCA CCATG ACCAG GGACA CGTCC ACGAG CACAG TCTAC ATGGA GCTGA GCAGC CTGAG ATCTG AGGAC ACGGC CGTGT ATTAC TGTGC GAGAG ATGGG TATGG CAGCT TCTCC CGGCT GATCC AGCTC TGGGG CCAGG GCACC CTGGT CACCG TCTCC TCA T0999H SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 598 NO: NO: 565 NO: 631 NO: 632 467 CAGGT QVQLI TCAGC QSGAK TGATT VKKPG CAGTC ASVKV CGGCG SCKAS CCAAA RYKFT GTGAA SYYMH GAAAC WVRQA CTGGC PGQGL GCCTC EWMGI TGTGA INPKS AGGTG GSTSY TCCTG AQKFQ CAAGG GRVTM CCTCT TRDTS CGGTA TSTVY CAAGT MELSS TCACC LRSED TCCTA TAVYY CTACA CARDG TGCAC YGSFS TGGGT RLIQL CCGAC WGQGT AGGCC LVTVS CCTGG S ACAAG GATTG GAGTG GATGG GCATC ATCAA CCCCA AGTCC GGCTC CACCT CTTAC GCCCA GAAAT TCCAG GGCAG AGTGA CCATG ACCAG AGACA CCTCT ACCTC CACCG TGTAC ATGGA ACTGT CCAGC CTGAG ATCCG AGGAC ACCGC CGTGT ACTAC TGTGC CAGAG ATGGC TACGG CAGCT TCTCC AGACT GATCC AGTTG TGGGG CCAGG GCACA CTGGT CACAG TGTCC TCT SEQ ID NO: 636. Consensus HCDR1. RYXFTSYY X is K or S Representing the T0201H VH CDR1 RYSFTSYY (SEQ ID NO: 462) in which the Ser at IMGT position 29 is retained or replaced by Lys. SEQ ID NO: 637. Consensus HCDR3. ARDGYGSX₁SRX₂X₃QL X1 is F or S. X2 is any amino acid. X3 is I or L. Representing the T0201H CDR3 ARDGYGSSSRCLQL (SEQ ID NO: 468) in which the Ser at IMGT position 111A is retained or replaced by Phe, the Cys at IMGT position 114 is retained or replaced by another amino acid residue, and the Leu at IMGT position 115 is retained or replaced by Ile. SEQ ID NO: 638. Consensus HCDR3. ARDGYGSX₁SRX₂X₃QL X1 is F or S. X2 is Leu or Val. X3 is I or L. Representing the T0201H CDR3 ARDGYGSSSRCLQL (SEQ ID NO: 468) in which the Ser at IMGT position 111A is retained or replaced by Phe, the Cys at IMGT position 114 is replaced by Leu or Val, and the Leu at IMGT position 115 is retained or replaced by Ile. SEQ ID NO: 639. Consensus HCDR3. ARDGY GSFSRXIQL X is Leu or Val. Representing the T0201H CDR3 ARDGY GSSSRCLQL (SEQ ID NO: 468) in which the Ser at IMGT position 111A is replaced by Phe, the Cys at IMGT position 114 is replaced by Leu or Val, and the Leu at IMGT position 115 is replaced by Ile.

TABLE S-11 Heavy chain sequences SEQ ID Nucleic GAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCC NO: 418 acid GGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAAC encoding ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCC N1280H- AAGAAATCCGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGC IgG4- TACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACC P K439E; AAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTG N1280 GTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTT coding CCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACC sequence CAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGC underlined. CCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAG GACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTG CAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCC ACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTT TACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTAC CCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTG GACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTC TCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCT SEQ ID N1280H- EVQLVESGGGFVQPGGSLRLSCAVSGFRENSYWMSWVRQAPGKGLEWVANINQDGSRKEYVASVKGRFTMSRDNA NO: 419 IgG4- KKSVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL P K439E VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYG amino acid PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNS sequence TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP SEQ ID Nucleic CAGGTTCAGCTGATTCAGTCCGGCGCCAAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCT NO: 420 acid CGGTACAAGTTCACCTCCTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATC encoding ATCAACCCCAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAGAGACACCTCT T0999H- ACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATGGC IgG4- TACGGCAGCTTCTCCAGACTGATCCAGTTGTGGGGCCAGGGCACACTGGTCACAGTGTCCTCTGCTTCCACCAAG P E356K; GGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTC T0999H AAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCA coding GCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAG sequence ACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCT underlined CCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGAC ACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACC TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC AACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTAC ACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCT TCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC TCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCC TGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT SEQ ID T0999H- QVQLIQSGAKVKKPGASVKVSCKASRYKFTSYYMHWVRQAPGQGLEWMGIINPKSGSTSYAQKFQGRVTMTRDTS NO: 421 IgG4- TSTVYMELSSLRSEDTAVYYCARDGYGSFSRLIQLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV P E356K KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGP amino acid PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNST sequence YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP SEQ ID Nucleic GAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCC NO: 423 acid GGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAAC encoding ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCC N1454H- AAGAAAGAGGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGC IgG4- TACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACC P K439E AAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTG GTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTT CCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACC CAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGC CCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAG GACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTG CAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCC ACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTT TACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTAC CCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTG GACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTC TCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCT SEQ ID N1454H- EVQLVESGGGFVQPGGSLRLSCAVSGFRENSYWMSWVRQAPGKGLEWVANINQDGSRKEYVASVKGRFTMSRDNA NO: 424 IgG4- KKEVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL P K439E VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYG amino acid PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNS sequence TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP SEQ ID Nucleic GAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCC NO: 425 acid GGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAAC encoding ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCC N1441H- GACAAGTCCGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGC IgG4- TACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACC P K439E AAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTG GTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTT CCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACC CAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGC CCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAG GACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTG CAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCC ACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTT TACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTAC CCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTG GACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTC TCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCT SEQ ID N1441H- EVQLVESGGGFVQPGGSLRLSCAVSGFRENSYWMSWVRQAPGKGLEWVANINQDGSRKEYVASVKGRFTMSRDNA NO: 426 IgG4- DKSVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL P K439E VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYG amino acid PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNS sequence TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP SEQ ID Nucleic GAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCC NO: 427 acid GGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAAC encoding ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCC N1442H- GAGAAGTCCGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGC IgG4- TACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACC P K439E AAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTG GTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTT CCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACC CAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGC CCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAG GACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTG CAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCC ACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTT TACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTAC CCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTG GACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTC TCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCT SEQ ID N1442H- EVQLVESGGGFVQPGGSLRLSCAVSGFRENSYWMSWVRQAPGKGLEWVANINQDGSRKEYVASVKGRFTMSRDNA NO: 428 IgG4- EKSVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL P K439E VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYG amino acid PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNS sequence TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP SEQ ID Nucleic CAGGTTCAGCTGATTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCT NO: 429 acid CGGTACAAGTTCACCTCCTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATC encoding ATCAACCCCAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAGAGACACCTCT T0736H- ACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATGGC IgG4- TACGGCAGCTTCTCCAGGCTGATCCAGTTGTGGGGACAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACCAAG P E356K GGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTC AAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCA GCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAG ACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCT CCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGAC ACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACC TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC AACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTAC ACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCT TCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC TCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCC TGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT SEQ ID T0736H- QVQLIQSGAEVKKPGASVKVSCKASRYKFTSYYMHWVRQAPGQGLEWMGIINPKSGSTSYAQKFQGRVTMTRDTS NO: 430 IgG4- TSTVYMELSSLRSEDTAVYYCARDGYGSFSRLIQLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV P E356K KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGP amino acid PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNST sequence YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP SEQ ID Nucleic CAGGTTCAGCTGATTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCC NO: 431 acid AGATACTCCTTCACCTCCTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATC encoding ATCAACCCCAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAGAGACACCTCT T0687H- ACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATGGC IgG4- TACGGCTCCTTCAGCAGAGTGATCCAGTTGTGGGGCCAGGGCACACTGGTCACAGTGTCCTCTGCTTCCACCAAG P E356K GGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTC AAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCA GCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAG ACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCT CCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGAC ACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACC TACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC AACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTAC ACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCT TCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGAC TCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCC TGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT SEQ ID T0687H- QVQLIQSGAEVKKPGASVKVSCKASRYSFTSYYMHWVRQAPGQGLEWMGIINPKSGSTSYAQKFQGRVTMTRDTS NO: 432 IgG4- TSTVYMELSSLRSEDTAVYYCARDGYGSFSRVIQLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV P E356K KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGP amino acid PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNST sequence YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP

Human Germline Gene Segments

TABLE S-12 Corresponding germline v and j gene seg- ments for antibody VH and VL domains Anti-FIX heavy chain V--J N128 IGHV3-7*01--IGHJ6*02 N183 IGHV3-48*02--IGHJ6*02 Anti FX heavy chain V--J T0200 IGHV1-46*03--IGHJ1*01 Common light chain V--J N0128L IGLV3-21*d01--IGLJ2*01

Common Light Chain Sequences

TABLE S-50A N0128 and N0325 VL domain sequences and CDRs VL amino VL nucleotide acid Ab VL LCDR1 LCDR2 LCDR3 sequence sequence N128L SEQ ID SEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 6 NO: 7 NO: 8 TCCTATGTGCTGACTC NO: 10 NIGRKS YDS QVWDGS AGCCACCCTCAGTGTC SYVLTQ SDHWV AGTGGCCCCAGGAGAG PPSVSV ACGGCCAGGATTACCT APGETA GTGGGGGAGACAACAT RITCGG TGGAAGGAAAAGTGTG DNIGRK TACTGGTACCAGCAGA SVYWYQ AGTCAGGCCAGGCCCC QKSGQA TGTGCTGGTCATCTAT PVLVIY TATGATAGCGACCGGC YDSDRP CCTCAGGGATCCCTGA SGIPER GCGATTCTCTGGGTCC FSGSNS AACTCTGGGAACACGG GNTATL CGACCCTGACCATCAG TISRVE CAGGGTCGAAGCCGGG AGDEAD GATGAGGCCGACTATT YYCQVW ACTGTCAGGTGTGGGA DGSSDH TGGAAGTAGTGATCAT WVFGGG TGGGTGTTCGGCGGAG TKLTVL GGACCAAGTTGACCGT CCTAG N325L SEQ ID SEQ ID SEQ ID SEQ ID NO: 415 SEQ ID NO: 6 NO: 7 NO: 8 TACGTGCTGACCCAGC NO: 416 CTCCTTCCGTGTCTGT YVLTQP TGCTCCTGGCGAGACA PSVSVA GCCAGAATCACCTGTG PGETAR GCGGCGATAACATCGG ITCGGD CCGGAAGTCCGTGTAC NIGRKS TGGTATCAGCAGAAGT VYWYQQ CCGGCCAGGCTCCTGT KSGQAP GCTGGTCATCTACTAC VLVIYY GACTCCGACCGGCCTT DSDRPS CTGGCATCCCTGAGAG GIPERF ATTCTCCGGCTCCAAC SGSNSG TCCGGCAATACCGCCA NTATLT CACTGACCATCTCCAG ISRVEA AGTGGAAGCTGGCGAC GDEADY GAGGCCGACTACTACT YCQVWD GCCAAGTGTGGGACGG GSSDHW CTCCTCTGACCACTGG VFGGGT GTTTTCGGCGGAGGCA KLTVL CCAAGCTGACAGTGCT G

TABLE S-50B N0128 and N0325 VL domain framework sequences Ab VL FR1 FR2 FR3 FR4 N128L SEQ ID SEQ ID SEQ ID SEQ ID NO: 136 NO:137 NO: 138 NO: 139 SYVLTQP VYWYQQK DRPSGIPER FGGGT PSVSVAP SGQAPVL FSGSNSGNT KLTVL GETARIT VIY ATLTISRVE CGGD AGDEADYYC N325L SEQ ID SEQ ID SEQ ID SEQ ID NO: 417 NO: 137 NO: 138 NO: 139 YVLTQP PSVSVA PGETAR ITCGGD

TABLE S-50C N0128 and N0325 light chain sequences SEQ ID N0128L- TCCTATGTGC TGACTCAGCC ACCCTCAGTG TCAGTGGCCC CAGGAGAGAC GGCCAGGATT NO: 404 IgL Coding ACCTGTGGGG GAGACAACAT TGGAAGGAAA AGTGTGTACT GGTACCAGCA GAAGTCAGGC nucleic acid CAGGCCCCTG TGCTGGTCAT CTATTATGAT AGCGACCGGC CCTCAGGGAT CCCTGAGCGA TTCTCTGGGT CCAACTCTGG GAACACGGCG ACCCTGACCA TCAGCAGGGT CGAAGCCGGG GATGAGGCCG ACTATTACTG TCAGGTGTGG GATGGAAGTA GTGATCATTG GGTGTTCGGC GGAGGGACCA AGTTGACCGT CCTAGGTCAG CCCAAGGCTG CCCCCTCGGT CACTCTGTTC CCACCCTCCT CTGAGGAGCT TCAAGCCAAC AAGGCCACAC TGGTGTGTCT CATAAGTGAC TTCTACCCGG GAGCCGTGAC AGTGGCCTGG AAGGCAGATA GCAGCCCCGT CAAGGCGGGA GTGGAGACCA CCACACCCTC CAAACAAAGC AACAACAAGT ACGCGGCCAG CAGCTACCTG AGCCTGACGC CTGAGCAGTG GAAGTCCCAC AAAAGCTACA GCTGCCAGGT CACGCATGAA GGGAGCACCG TGGAGAAGAC AGTGGCCCCT ACAGAATGTT CA SEQ ID N0128L light SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSN NO: 405 chain amino SGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQAN acid sequence KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSC (mature) QVTHEGSTVEKTVAPTECS SEQ ID N0325L-IgL TACGTGCTGACCCAGCCTCCTTCCGTGTCTGTTGCTCCTGGCGAGACAGCCAGAATCACCTGTGG NO: 413 coding nucleic CGGCGATAACATCGGCCGGAAGTCCGTGTACTGGTATCAGCAGAAGTCCGGCCAGGCTCCTGTGC acid; N0325L TGGTCATCTACTACGACTCCGACCGGCCTTCTGGCATCCCTGAGAGATTCTCCGGCTCCAACTCC coding GGCAATACCGCCACACTGACCATCTCCAGAGTGGAAGCTGGCGACGAGGCCGACTACTACTGCCA sequence AGTGTGGGACGGCTCCTCTGACCACTGGGTTTTCGGCGGAGGCACCAAGCTGACAGTGCTGGGAC underlined AACCTAAGGCCGCTCCTTCTGTGACCCTGTTTCCTCCATCCTCCGAGGAACTGCAGGCCAACAAG GCTACCCTCGTGTGCCTGATCTCCGACTTTTACCCTGGCGCTGTGACCGTGGCCTGGAAGGCTGA TAGTTCTCCTGTGAAGGCCGGCGTGGAAACCACCACACCTTCCAAGCAGTCCAACAACAAATACG CCGCCTCCTCCTACCTGTCTCTGACCCCTGAACAGTGGAAGTCCCACAAGTCCTACTCTTGCCAA GTGACCCACGAGGGCTCCACCGTGGAAAAGACAGTGGCTCCTACCGAGTGCTCC SEQ ID N0325L light YVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNS NO: 414 chain amino GNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK acid sequence ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQ (mature) VTHEGSTVEKTVAPTECS Recombinant expression of bispecific antibody using common light chain N0128L with its native human Igλ leader sequence (v3-21 leader peptide MAWTALLLGLLSHCTGSVT SEQ ID NO: 519) resulted in clipping of the N terminal Ser to produce antibody in which the VL domain was identical to the sequence shown herein for N0325 VL domain. For use with alternative leader sequences in which the mature light chain polypeptide is produced by cleavage after the Ser, the light chain 0325 was generated in order to achieve the same mature product. 0325 omits the N terminal Ser residue of 0128L.

Constant Regions

TABLE S-100 Antibody constant region sequences IgG4 PE human SEQ ID NO: 143 heavy chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT constant region FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK IgG4 human heavy SEQ ID NO: 144 chain constant ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT region with knobs- FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP into-holes PCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN mutations and WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL hinge mutation. PSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVE Type a (IgG4ra) WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK IgG4 human heavy SEQ ID NO: 145 chain constant ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT region with FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP knobs-into- PCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN holes mutations WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL and hinge mutation. PSSIEKTISKAKGQPREPQVYTLPPSQCEMTKNQVSLSCAVKGFYPSDIAVE Type b (IgG4yb) WESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK IgG4 human heavy SEQ ID NO: 409 chain constant ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT region with P FPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGP (hinge) mutation PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN and K439E WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEAL HNHYTQESLSLSP IgG4 human heavy SEQ ID NO: 410 chain constant ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT region with P FPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGP (hinge) mutation PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN and E356K WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSP IgG4-P K439E SEQ ID NO: 411 encoding nucleic GCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCA acid CCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGA GCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACC TTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGA CCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCA CAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCT CCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTC TGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGT GACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAAT TGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGG AACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCA GGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTG CCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAAC CCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGT GTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAA TGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGC TGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTC CCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTG CACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCT IgG4-P E356K SEQ ID NO: 412 encoding GCTTCCACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCA nucleic acid CCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGA GCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACC TTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGA CCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCA CAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCT CCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTC TGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGT GACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAAT TGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGG AACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCA GGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTG CCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAAC CCCAGGTTTACACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGT GTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAA TGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGC TGGACTCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTC CCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTG CACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT Nucleic acid SEQ ID NO: 633 encoding IgL GGACAACCTAAGGCCGCTCCTTCTGTGACCCTGTTTCCTCCATCCTCCGAGG human lambda AACTGCAGGCCAACAAGGCTACCCTCGTGTGCCTGATCTCCGACTTTTACCC light chain TGGCGCTGTGACCGTGGCCTGGAAGGCTGATAGTTCTCCTGTGAAGGCCGGC constant GTGGAAACCACCACACCTTCCAAGCAGTCCAACAACAAATACGCCGCCTCCT region CCTACCTGTCTCTGACCCCTGAACAGTGGAAGTCCCACAAGTCCTACTCTTG CCAAGTGACCCACGAGGGCTCCACCGTGGAAAAGACAGTGGCTCCTACCGAG TGCTCC Human lambda SEQ ID NO: 146 light chain GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG constant region VETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTE CS Human kappa SEQ ID NO: 147 light chain KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN constant region SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

TABLE N N128H Alanine Scanning Mutants CDR1 G F T F N S Y W (GFTFNSYW) N400H N401H N402H N403H N404H N405H N406H N407H CDR2 I N Q D G S E K (INQDGSEK) N408H N409H N410H N411H N412H N413H N414H N415H CDR3 A R E G Y S S S S Y Y G M D V (AREGYSSS N416H N417H N418H N419H N420H N421H N422H N423H N424H N425H N426H N427H N428H N429H SYYGMDV) N128H CDR3 Mutants CDR3 A C D E F G H I K L M N P Q R S T V W Y AREGYSSS N420H N467H N468H N469H N470H N471H N472H N473H N474H N475H N476H N477H N478H N479H N480H N128H N481H N482H N483H N484H SYYGMDV AREGYSSS N421H N485H N486H N487H N488H N489H N490H N491H N492H N493H N494H N495H N496H N497H N498H N128H N499H N500H N501H N502H SYYGMDV AREGYSSS N422H N430H N431H N432H N433H N434H N435H N436H N437H N438H N439H N440H N441H N442H N443H N128H N444H N445H N446H N447H SYYGMDV AREGYSSS N423H N448H N449H N450H N451H N452H N453H N454H N455H N456H N457H N458H N459H N460H N461H N128H N462H N463H N464H N465H SYYGMDV AREGYSSS N424H N542H N543H N544H N545H N546H N547H N548H N549H N550H N551H N552H N553H N554H N555H N559H N556H N557H N558H N128H SYYGMDV AREGYSSS N425H N561H N562H N563H N564H N565H N566H N567H N568H N569H N570H N571H N572H N573H N574H N578H N575H N576H N577H N128H SYYGMDV AREGYSSS N426H N579H N580H N581H N582H N128H N583H N584H N585H N586H N587H N588H N589H N590H N591H N592H N593H N594H N595H N596H SYYGMDV AREGYSSS N427H N597H N598H N599H N600H N601H N602H N603H N604H N605H N128H N606H N607H N608H N609H N610H N611H N612H N613H N614H SYYGMDV AREGYSSS N428H N615H N128H N616H N617H N618H N619H N620H N621H N622H N623H N624H N625H N626H N627H N628H N629H N630H N631H N632H SYYGMDV AREGYSSS N429H N633H N634H N635H N636H N637H N638H N639H N640H N641H N642H N643H N644H N645H N646H N647H N648H N128H N649H N650H SYYGMDV AREGYSSS N128H N651H N652H N653H N654H N655H N656H N657H N658H N659H N660H N661H N662H N663H N664H N665H N666H N667H N668H N669H SYYGMDV AREGYSSS N416H N670H N671H N672H N673H N674H N675H N676H N677H N678H N679H N680H N681H N682H N128H N683H N684H N685H N686H N687H SYYGMDV AREGYSSS N417H N689H N690H N128H N691H N692H N693H N694H N695H N696H N697H N698H N699H N700H N701H N702H N703H N704H N705H N706H SYYGMDV AREGYSSS N418H N707H N708H N709H N710H N128H N711H N712H N713H N714H N715H N716H N717H N718H N719H N720H N721H N722H N723H N724H SYYGMDV AREGYSSS N419H N725H N726H N727H N728H N729H N730H N731H N732H N733H N734H N735H N736H N737H N738H N739H N740H N741H N742H N128H SYYGMDV N436H CDR3 Mutants CDR3 A C D E F G H I K L M N P Q R S T V W Y AREGYSSI N503H N504H N505H N506H N507H N508H N509H N510H N511H N512H N513H N514H N515H N516H N517H N436H N518H N519H N520H N521H SYYGMDV AREGYSSI N522H N523H N524H N525H N526H N527H N528H N529H N530H N531H N532H N533H N534H N535H N536H N436H N537H N538H N539H N540H SYYGMDV N436H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW N819H N820H N821H N822H N436H N823H N824H N825H N826H N827H N828H N829H N830H N831H N832H N833H N834H N835H N836H GFTFNSYW N837H N838H N839H N436H N840H N841H N842H N843H N844H N845H N846H N847H N848H N849H N850H N851H N852H N853H N854H GFTFNSYW N856H N857H N858H N859H N860H N861H N862H N863H N864H N865H N866H N867H N868H N869H N870H N436H N871H N872H N873H GFTFNSYW N874H N875H N876H N436H N877H N878H N879H N880H N881H N882H N883H N884H N885H N886H N887H N888H N889H N890H N891H GFTFNSYW N892H N893H N894H N895H N896H N897H N898H N899H N900H N901H N436H N902H N903H N904H N905H N906H N907H N908H N909H GFTFNSYW N910H N911H N912H N913H N914H N915H N916H N917H N918H N919H N920H N921H N922H N923H N436H N924H N925H N926H N927H GFTFNSYW N928H N929H N930H N931H N932H N933H N934H N935H N936H N937H N938H N939H N940H N941H N942H N943H N944H N945H N436H GFTFNSYW N946H N947H N948H N949H N950H N951H N952H N953H N954H N955H N956H N957 N958H N959H N960H N961H N962H N436H N963H N436H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S T V W Y INQDGSEK N964H N965H N966H N967H N968H N969H N970H N436H N971H N972H N973H N974H N975H N976H N977H N978H N979H N980H N981H N982H INQDGSEK N983H N984H N985H N986H N987H N988H N989H N990H N991H N992H N993H N436H N994H N995H N996H N997H N998H N999H N1000H N1001H INQDGSEK N1002H N1003H N1004H N1005H N1006H N1007H N1008H N1009H N1010H N1011H N1012H N1013H N1014H N436H N1015H N1016H N1017H N1018H N1019H N1020H INQDGSEK N1021H N1022H N436H N1023H N1024H N1025H N1026H N1027H N1028H N1029H N1030H N1031H N1032H N1033H N1034H N1035H N1036H N1037H N1038H N1039H INQDGSEK N1040H N1041H N1042H N1043H N1044H N436H N1045H N1046H N1047H N1048H N1049H N1050H N1051H N1052H N1053H N1054H N1055H N1056H N1057H N1058H INQDGSEK N1059H N1060H N1061H N1062H N1063H N1064H N1065H N1066H N1067H N1068H N1069H N1070H N1071H N1072H N1073H N436H N1074H N1075H N1076H N1077H INQDGSEK N1078H N1079H N1080H N436H N1081H N1082H N1083H N1084H N1085H N1086H N1087H N1088H N1089H N1090H N1091H N1092H N1093H N1094H N1095H N1096H INQDGSEK N1097H N1098H N1099H N1100H N1101H N1102H N1103H N1104H N436H N1105H N1106H N1107H N1108H N1109H N1110H N1111H N1112H N1113H N1114H N1115H N511H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S T V W Y INQDGSEK N1116H N1117H N1118H INQDGSEK INQDGSEK N1119H INQDGSEK N1120H N1121H N511H N1122H N1123H N1124H N1125H N1126H N1127H N1128H N1129H N1130H N1131H N1132H N1133H N1134H N1135H N1136H N1137H N1138H INQDGSEK N1139H INQDGSEK N1140H N1141H N1142H N1143H N1144H N1145H N1146H N1147H N1148H N1149H N1150H N1151H N1152H N1153H N1154H N511H N1155H N1156H N1157H N1158H INQDGSEK N1159H N1160H N1161H N511H N1162H N1163H N1164H N1165H N1166H N1167H N1168H N1169H N1170H N1171H N1172H N1173H N1174H N1175H N1176H N1177H INQDGSEK Selected N436H CDR1 Mutants (batch 1) CDR1 A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW N825H N832H N833H GFTFNSWY N849H GFTFNSWY N863H N866H GFTFNSYW N875H N878H N889H GFTFNSYW GFTFNSYW N917H N920H N921H N925H GFTFNSYW N934H N936H N937H N939H N940H N941H N942H N943H N944H N945H GFTFNSYW N946H N947H N948H N949H N950H N951H N952H N953H N954H N955H N956H N957H N963H N511H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW N1178H N1179H N1180H GFTFNSYW N1181H GFTFNSYW N1182H N1183H GFTFNSYW N1211H N1212H N1213H GFTFNSYW GFTFNSYW N1184H N1185H N1186H N1187H GFTFNSYW N1188H N1189H N1190H N1191H N1192H N1193H N1194H N1195H N1196H N1197H GFTFNSYW N1198H N1199H N1200H N1201H N1202H N1203H N1204H N1205H N1206H N1207H N1208H N1209H N1210H N1172H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW N1214H N1215H N1216H GFTFNSYW N1217H GFTFNSYW N1218H N1219H GFTFNSYW N1247H N1248H N1249H GFTFNSYW GFTFNSYW N1220H N1221H N1222H N1223H GFTFNSYW N1224H N1225H N1226H N1227H N1228H N1229H N1230H N1231H N1232H N1233H GFTFNSYW N1234H N1235H N1236H N1237H N1238H N1239H N1240H N1241H N1242H N1243H N1244H N1245H N1246H Selected N436H CDR1 Mutants (batch 2) CDR1 A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW GFTFNSYW GFTFNSYW N869H N872H N873H GFTFNSYW N877H N886H N888H N889H N891H GFTFNSYW N892H N893H N894H N895H N896H N897H N898H N899H N900H N901H N902H N903H N904H N905H N906H N907H N908H N909H GFTFNSYW N915H N923H N926H GFTFNSYW N937H GFTFNSYW N511H CDR1 Mutants A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW GFTFNSYW GFTFNSYW N1250H N1251H N1252H GFTFNSYW N1253H N1254H N1255H N1256H N1257H GFTFNSYW N1258H N1259H N1260H N1261H N1262H N1263H N1264H N1265H N1266H N1267H N1268H N1269H N1270H N1271H N1272H N1273H N1274H N1275H GFTFNSYW N1276H N1277H N1278H GFTFNSYW N1279H GFTFNSYW N1172H CDR1 Mutants A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW GFTFNSYW GFTFNSYW N1280H N1281H N1282H GFTFNSYW N1283H N1284H N1285H N1286H N1287H GFTFNSYW N1288H N1289H N1290H N1291H N1292H N1293H N1294H N1295H N1296H N1297H N1298H N1299H N1300H N1301H N1302H N1303H N1304H N1305H GFTFNSYW N1306H N1307H N1308H GFTFNSYW N1309H GFTFNSYW N1280H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S T V W Y INQDGSRK N1310H N1311H N1280H N1312H N1313H N1314H N1315H N1316H N1317H N1318H N1319H N1320H N1321H N1322H N1323H N1324H N1325H N1326H N1327H N1328H INQDGSRK N1329H N1330H N1331H N1332H N1333H N1334H N1335H N1336H N1337H N1338H N1339H N1340H N1341H N1342H N1343H N1280H N1344H N1345H N1346H N1347H N1280H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R S T V W Y GFRFNSYW N1367H N1368H N1369H N1370H N1371H N1372H N1373H N1374H N1375H N1376H N1377H N1378H N1379H N1380H N1381H N1382H N1383H N1384H N1385H N1280H N1280H CDR1 Double Mutants (all including Arg29Lys, with Tyr37 mutated as shown) CDR1 A C D F F G H I K L M N P Q R S T V W Y GFKFNSYW N1348H N1349H N1350H N1351H N1352H N1353H N1354H N1355H N1356H N1357H N1358H N1359H N1360H N1361H N1362H N1363H N1364H N1365H N1366H T0201H CDR3 Mutants CDR3 A C D E F G H I K L M N P Q R S T V W Y ARDGYGS T400H T401H T402H T403H T404H T405H T406H T407H T408H T409H T410H T411H T412H T413H T414H T415H T416H T417H T418H SSRCLQL ARDGYGS T419H T420H T421H T422H T423H T424H T425H T426H T427H T428H T429H T430H T431H T432H T433H T434H T435H T436H T437H SSRCLQL ARDGYGS T438H T439H T440H T441H T442H T443H T444H T445H T446H T447H T448H T449H T450H T451H T452H T453H T454H T455H T456H SSRCLQL ARDGYGS T457H T458H T459H T460H T461H T462H T463H T464H T465H T466H T467H T468H T469H T470H T471H T472H T473H T474H T475H SSRCLQL ARDGYGS T476H T477H T478H T479H T480H T481H T482H T483H T484H T485H T486H T487H T488H T489H T490H T491H T492H T493H T494H SSRCLQL ARDGYGS T495H T496H T497H T498H T499H T500H T501H T502H T503H T504H T505H T506H T507H T508H T509H T510H T511H T512H T513H SSRCLQL ARDGYGS T514H T515H T516H T517H T518H T519H T520H T521H T522H T523H T524H T525H T526H T527H T528H T529H T530H T531H T532H SSRCLQL ARDGYGS T533H T534H T535H T536H T537H T538H T539H T540H T541H T542H T543H T544H T545H T546H T547H T548H T549H T550H T551H SSRCLQL ARDGYGS T552H T553H T554H T555H T556H T557H T558H T559H T560H T561H T562H T563H T564H T565H T566H T567H T568H T569H T570H SSRCLQL ARDGYGS T571H T572H T573H T574H T575H T576H T577H T578H T579H T580H T581H T582H T583H T584H T585H T586H T587H T588H T589H SSRCLQL ARDGYGS T590H T591H T592H T593H T594H T595H T596H T597H T598H T599H T600H T601H T602H T603H T604H T605H T606H T607H T608H SSRCLQL ARDGYGS T609H T610H T611H T612H T613H T614H T615H T616H T617H T618H T619H T620H T621H T622H T623H T624H T625H T626H T627H SSRCLQL ARDGYGS T628H T629H T630H T631H T632H T633H T634H T635H T636H T637H T638H T639H T640H T641H T642H T643H T644H T645H T646H SSRCLQL ARDGYGS T647H T648H T649H T650H T651H T652H T653H T654H T655H T656H T657H T658H T659H T660H T661H T662H T663H T664H T665H SSRCLQL

TABLE T VH domain CDR1 CDR2 CDR3 T0201H RYSFTSYY INPKSGST ARDGYGSSSRCLQL CDR3 compound mutation T666H RYSFTSYY INPKSGST ARDGYGSSSRIIQL T667H RYSFTSYY INPKSGST ARDGYGSSSRLIQL T668H RYSFTSYY INPKSGST ARDGYGSSSRQIQL T669H RYSFTSYY INPKSGST ARDGYGSSSRILML T670H RYSFTSYY INPKSGST ARDGYGSSSRLLML T671H RYSFTSYY INPKSGST ARDGYGSSSRQLML T672H RYSFTSYY INPKSGST ARDGYGSSSRIIML T673H RYSFTSYY INPKSGST ARDGYGSSSRLIML T674H RYSFTSYY INPKSGST ARDGYGSSSRQIML T675H RYSFTSYY INPKSGST ARDGYGSSSRVIQL T676H RYSFTSYY INPKSGST ARDGYGSSSRVLML T677H RYSFTSYY INPKSGST ARDGYGSSSRVIML T678H RYSFTSYY INPKSGST ARDGYGSFSRIIQL T679H RYSFTSYY INPKSGST ARDGYGSFSRILML T680H RYSFTSYY INPKSGST ARDGYGSFSRIIML T681H RYSFTSYY INPKSGST ARDGYGSFSRLIQL T682H RYSFTSYY INPKSGST ARDGYGSFSRLLML T683H RYSFTSYY INPKSGST ARDGYGSFSRLIML T684H RYSFTSYY INPKSGST ARDGYGSFSRQIQL T685H RYSFTSYY INPKSGST ARDGYGSFSRQLML T686H RYSFTSYY INPKSGST ARDGYGSFSRQIML T687H RYSFTSYY INPKSGST ARDGYGSFSRVIQL T688H RYSFTSYY INPKSGST ARDGYGSFSRVLML T689H RYSFTSYY INPKSGST ARDGYGSFSRVIML T0681H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R S T V W Y RYSFTSYY T690H T691H T692H T693H T694H T695H T696H T697H T698H T699H T700H T701H T702H T703H T704H T705H T706H T707H T708H RYSFTSYY T709H T710H T711H T712H T713H T714H T715H T716H T717H T718H T719H T720H T721H T722H T723H T724H T725H T726H T727H RYSFTSYY T728H T729H T730H T731H T732H T733H T734H T735H T736H T737H T738H T739H T740H T741H T742H T743H T744H T745H T746H RYSFTSYY T747H T748H T749H T750H T751H T752H T753H T754H T755H T756H T757H T758H T759H T760H T761H T762H T763H T764H T765H RYSFTSYY T766H T767H T768H T769H T770H T771H T772H T773H T774H T775H T776H T777H T778H T779H T780H T781H T782H T783H T784H RYSFTSYY T785H T786H T787H T788H T789H T790H T791H T792H T793H T794H T795H T796H T797H T798H T799H T800H T801H T802H T803H RYSFTSYY T804H T805H T806H T807H T808H T809H T810H T811H T812H T813H T814H T815H T816H T817H T818H T819H T820H T821H T822H RYSFTSYY T823H T824H T825H T826H T827H T828H T829H T830H T831H T832H T833H T834H T835H T836H T837H T838H T839H T840H T841H T0681H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S T V W Y INPKSGST T842H T843H T844H T845H T846H T847H T848H T849H T850H T851H T852H T853H T854H T855H T856H T857H T858H T859H T860H INPKSGST T861H T862H T863H T864H T865H T866H T867H T868H T869H T870H T871H T872H T873H T874H T875H T876H T877H T878H T879H INPKSGST T880H T881H T882H T883H T884H T885H T886H T887H T888H T889H T890H T891H T892H T893H T894H T895H T896H T897H T898H INPKSGST T899H T900H T901H T902H T903H T904H T905H T906H T907H T908H T909H T910H T911H T912H T913H T914H T915H T916H T917H INPKSGST T918H T919H T920H T921H T922H T923H T924H T925H T926H T927H T928H T929H T930H T931H T932H T933H T934H T935H T936H INPKSGST T937H T938H T939H T940H T941H T942H T943H T944H T945H T946H T947H T948H T949H T950H T951H T952H T953H T954H T955H INPKSGST T956H T957H T958H T959H T960H T961H T962H T963H T964H T965H T966H T967H T968H T969H T970H T971H T972H T973H T974H INPKSGST T975H T976H T977H T978H T979H T980H T981H T982H T983H T984H T985H T986H T987H T988H T989H T990H T991H T992H T993H CDR2 mutations T678H T850H T925H T926H T951H T958H T989H T990H T678H INPKSGST LNPKSGST INPKIGST INPKKGST INPKSSST INPKSGDT INPKSGSR INPKSGSS CDR1 T678H RYSFTSYY T678H T1015H T1022H T1029H T1036H T1043H T1050H T1057H mutations T713H RFSFTSYY T1009H T1016H T1023H T1030H T1037H T1044H T1051H T1058H T734H RYHFTSYY T1010H T1017H T1024H T1031H T1038H T1045H T1052H T1059H T736H RYKFTSYY T1011H T1018H T1025H T1032H T1039H T1046H T1053H T1060H T742H RYRFTSYY T1012H T1019H T1026H T1033H T1040H T1047H T1054H T1061H T774H RYSFKSYY T1013H T1020H T1027H T1034H T1041H T1048H T1055H T1062H T785H RYSFTAYY T1014H T1021H T1028H T1035H T1042H T1049H T1056H T1063H CDR2 mutations T681H T850H T925H T926H T951H T958H T989H T990H T681H INPKSGST LNPKSGST INPKIGST INPKKGST INPKSSST INPKSGDT INPKSGSR INPKSGSS CDR1 T681H RYSFTSYY T681H T850H T925H T926H T951H T958H T989H T990H mutations T713H RFSFTSYY T713H T1064H T1070H T1076H T1082H T1088H T1094H T1100H T734H RYHFTSYY T734H T1065H T1071H T1077H T1083H T1089H T1095H T1101H T736H RYKFTSYY T736H T1066H T1072H T1078H T1084H T1090H T1096H T1102H T742H RYRFTSYY T742H T1067H T1073H T1079H T1085H T1091H T1097H T1103H T774H RYSFKSYY T774H T1068H T1074H T1080H T1086H T1092H T1098H T1104H T785H RYSFTAYY T785H T1069H T1075H T1081H T1087H T1093H T1099H T1105H CDR2 mutations T687H T850H T925H T926H T951H T958H T989H T990H T687H INPKSGST LNPKSGST INPKIGST INPKKGST INPKSSST INPKSGDT INPKSGSR INPKSGSS CDR1 T687H RYSFTSYY T687H T1113H T1120H T1127H T1134H T1141H T1148H T1155H mutations T713H RFSFTSYY T1107H T1114H T1121H T1128H T1135H T1142H T1149H T1156H T734H RYHFTSYY T1108H T1115H T1122H T1129H T1136H T1143H T1150H T1157H T736H RYKFTSYY T1109H T1116H T1123H T1130H T1137H T1144H T1151H T1158H T742H RYRFTSYY T1110H T1117H T1124H T1131H T1138H T1145H T1152H T1159H T774H RYSFKSYY T1111H T1118H T1125H T1132H T1139H T1146H T1153H T1160H T785H RYSFTAYY T1112H T1119H T1126H T1133H T1140H T1147H T1154H T1161H 

What is claimed is:
 1. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the first VH domain comprises a set of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408, and/or wherein the first VH domain is at least 95% identical to the N1280H VH domain at the amino acid sequence level; the second VH domain is at least 95% identical to the T0201H VH domain SEQ ID NO: 470 at the amino acid sequence level, and the first VL domain and the second VL domain each comprise a set of LCDRs comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences defined wherein LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7 and LCDR3 is SEQ ID NO: 8, and/or wherein the first VL domain and the second VL domain are at least 95% identical to the 0128L VL domain SEQ ID NO: 10 at the amino acid sequence level.
 2. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the first VH domain is a product of recombination of human immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is IGHV3-7 (e.g., VH3-7*01) and the j gene segment is IGHJ6 (e.g., JH6*02), the second VH domain is a product of recombination of human immunoglobulin heavy chain v, d and j gene segments, wherein the v gene segment is IGHV1-46 (e.g., VH1-46*03) and the j gene segment is IGHJ1 (e.g., JH1*01), and optionally wherein the d gene segment is IGHD6-6 (e.g., DH6-6*01), and the first VL domain and the second VL domain are both products of recombination of human immunoglobulin light chain v and j gene segments, wherein the v gene segment is IGLV3-21 (e.g., VL3-21*d01) and the j gene segment is IGLJ2 (e.g., JL2*01) or IGLJ3 (e.g., JL3*02).
 3. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, and a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the first VH domain has at least 95% amino acid sequence identity with the N1280H VH domain SEQ ID NO: 443, the second VH domain has at least 95% amino acid sequence identity with the 10201H VH domain SEQ ID NO: 470, and the first VL domain and the second VL domain each have at least 95% amino acid sequence identity with the 0128L VL domain SEQ ID NO:
 10. 4. Bispecific antibody according to any preceding claim, wherein the first VH domain comprises a set of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO:
 408. 5. Bispecific antibody according to claim 4, wherein the first VH domain comprises HCDR1 SEQ ID NO:
 441. 6. Bispecific antibody according to claim 4 or claim 5, wherein the first VH domain comprises HCDR2 SEQ ID NO:
 634. 7. Bispecific antibody according to any of claims 4 to 6, wherein the first VH domain comprises HCDR2 SEQ ID NO:
 436. 8. Bispecific antibody according to any of claims 4 to 7, wherein the first VH domain comprises HCDR3 SEQ ID NO:
 635. 9. Bispecific antibody according to any of claims 4 to 8, wherein the first VH domain comprises HCDR3 SEQ ID NO:
 433. 10. Bispecific antibody according to any preceding claim, wherein the first VH domain has at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to N1280H.
 11. Bispecific antibody according to any preceding claim, wherein the first VH domain comprises a set of N1280H HCDRs comprising N1280H HCDR1 SEQ ID NO: 441, N1280H HCDR2 SEQ ID NO: 436 and N1280H HCDR3 SEQ ID NO:
 433. 12. Bispecific antibody according to claim 10 or claim 11, wherein the first VH domain is the N1280H VH domain SEQ ID NO:
 443. 13. Bispecific antibody according to any of claims 1 to 11, wherein the first VH domain is the N1441H VH domain SEQ ID NO:
 456. 14. Bispecific antibody according to any preceding claim, wherein the second VH domain comprises an HCDR1 which is the T0201H HCDR1 SEQ ID NO: 462, an HCDR2 which is the T0201H HCDR2 SEQ ID NO: 467, and/or an HCDR3 which is the T0201H HCDR3 SEQ ID NO:
 468. 15. Bispecific antibody according to any preceding claim wherein the second VH domain comprises HCDR1 SEQ ID NO: 636 or SEQ ID NO: 598; wherein the second VH domain comprises HCDR2 SEQ ID NO: 467; and/or wherein the second VH domain comprises HCDR3 SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639 or SEQ ID NO:
 565. 16. Bispecific antibody according to any preceding claim, wherein the second VH domain comprises SEQ ID NO:
 632. 17. Bispecific antibody according to any preceding claim, wherein the first VL domain and the second VL domain each have at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with 0128L SEQ ID NO:
 10. 18. Bispecific antibody according to any preceding claim, wherein the first VL domain and the second VL domain each comprise a set of 0128L CDRs comprising 0128L LCDR1 SEQ ID NO: 6, 0128L LCDR2 SEQ ID NO: 7 and 0128L LCDR3 SEQ ID NO:
 8. 19. Bispecific antibody according to any preceding claim, wherein the first VL domain and the second VL domain are identical in amino acid sequence.
 20. Bispecific antibody according to claim 19, wherein the first VL domain and the second VL domain comprise the 0325L amino acid sequence SEQ ID NO:
 416. 21. Bispecific antibody according to any preceding claim, wherein each heavy-light chain pair further comprises a CL constant domain paired with a CH1 domain.
 22. Bispecific antibody according to any preceding claim, wherein the heavy-light chain pairs comprise a common light chain.
 23. Bispecific antibody according to claim 22, wherein the common light chain comprises the CL amino acid sequence SEQ ID NO: 146 of the 0128L light chain.
 24. Bispecific antibody according to claim 23, wherein the common light chain is the 0325L light chain SEQ ID NO:
 414. 25. Bispecific antibody according to any preceding claim, wherein the heavy chain of each heavy-light chain comprises a heavy chain constant region and wherein the first and second heavy-light chain pairs associate to form tetrameric immunoglobulin through dimerisation of the heavy chain constant regions.
 26. Bispecific antibody according to claim 25, wherein the heavy chain constant region of the first heavy-light chain pair comprises a different amino acid sequence from the heavy chain constant region of the second heavy-light chain pair, wherein the different amino acid sequences are engineered to promote heterodimerisation of the heavy chain constant regions.
 27. Bispecific antibody according to claim 26, wherein the heavy chain constant regions comprise knobs-into-holes mutations or charge pair mutations.
 28. Bispecific antibody according to claim 26, wherein the heavy chain constant region of one (e.g., the first) heavy-light chain pair is a human IgG4 constant region comprising substitution K439E and wherein the heavy chain constant region of the other (e.g., the second) heavy-light chain pair is an IgG4 region comprising substitution E356K, wherein constant region numbering is according to the EU numbering system.
 29. Bispecific antibody according to any of claims 25 to 28, wherein the heavy chain constant region of one or both heavy-light chain pairs is a human IgG4 constant region comprising substitution S228P, wherein constant region numbering is according to the EU numbering system.
 30. Bispecific antibody according to any of claims 25 to 29, wherein the heavy chain constant region of one (e.g., the first) heavy-light chain pair comprises SEQ ID NO: 409 and the heavy chain constant region of the other (e.g., the second) heavy-light chain pair comprises SEQ ID NO:
 410. 31. Bispecific antibody according to any of claims 25 to 30, comprising a first heavy chain comprising a first VH domain amino acid sequence SEQ ID NO: 443 or SEQ ID NO: 456, a second heavy chain comprising a second VH domain amino acid sequence SEQ ID NO: 632, and a common light chain comprising a VL domain amino acid sequence SEQ ID NO:
 416. 32. Bispecific antibody according to any of claims 25 to 31, comprising a first heavy chain comprising amino acid sequence SEQ ID NO: 419, a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and a common light chain comprising amino acid sequence SEQ ID NO:
 414. 33. Bispecific antibody according to any of claims 25 to 32, comprising a first heavy chain comprising amino acid sequence SEQ ID NO: 426 a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and a common light chain comprising amino acid sequence SEQ ID NO:
 414. 34. Bispecific antibody according to any of claims 1 to 27, wherein the antibody is human IgG.
 35. Bispecific antibody according to claim 34, wherein the antibody is human IgG4.
 36. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1280H VH domain SEQ ID NO: 443, and a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the 10999H VH domain SEQ ID NO: 632, and wherein the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO:
 414. 37. Bispecific antibody that binds FIXa and FX and catalyses FIXa-mediated activation of FX, wherein the antibody comprises two immunoglobulin heavy-light chain pairs, wherein a first heavy-light chain pair comprises a FIXa binding Fv region comprising a first VH domain paired with a first VL domain, wherein the first VH domain is at least 98% identical in amino acid sequence to the N1441H VH domain SEQ ID NO: 456, and a second heavy-light chain pair comprises a FX binding Fv region comprising a second VH domain paired with a second VL domain, wherein the second VH domain is at least 98% identical in amino acid sequence to the 10999H VH domain SEQ ID NO: 632, and wherein the first and second heavy-light chain pairs each comprise a common light chain comprising the 0325L light chain amino acid sequence SEQ ID NO:
 414. 38. Bispecific antibody according to any preceding claim, which reduces the coagulation time of FVIII-deficient human blood plasma to 22-28 seconds in an aPTT assay.
 39. Bispecific antibody according to any preceding claim, which enhances the FIXa-mediated activation of FX to FXa to at least the same extent as emicizumab.
 40. Bispecific antibody according to claim 38, wherein said coagulation time or FIXa-mediated activation is as determined at an antibody concentration of 0.1 mg/ml, 0.3 mg/ml or 0.5 mg/ml at 37 degrees C.
 41. Bispecific antibody according to any preceding claim, wherein the antibody has an EC50 for Cmax in a fluorometric thrombin generation assay (TGA) that is within 10% of or is lower than the Cmax EC50 of emicizumab in said assay, and/or wherein the antibody generates a maximal response of Cmax between 200 and 450 nM thrombin in a fluorometric TGA.
 42. Bispecific antibody according to claim 41, wherein the antibody has an EC50 of less than 50 nM for Cmax in a fluorometric TGA.
 43. Bispecific antibody according to claim 42, which has an EC50 of less than 10 nM for Cmax in a fluorimetric TGA.
 44. Bispecific antibody according to any preceding claim, wherein the maximal response of Cmax is between 250 nM and 400 nM.
 45. Anti-FIXa antibody comprising two copies of the first heavy-light chain pair as defined in any preceding claim.
 46. Anti-FX antibody comprising two copies of the second heavy-light chain pair as defined in any of claims 1 to
 44. 47. Isolated nucleic acid encoding an antibody according to any preceding claim.
 48. A host cell in vitro comprising recombinant DNA encoding an antibody heavy chain comprising a first VH domain as defined in any of claims 1 to 44, an antibody heavy chain comprising a second VH domain as defined in any of claims 1 to 44, and/or an antibody light chain comprising a first or second VL domain as defined in any of claims 1 to
 44. 49. A host cell according to claim 48 comprising recombinant DNA encoding a first heavy chain comprising amino acid sequence SEQ ID NO: 419 or SEQ ID NO: 426, a second heavy chain comprising amino acid sequence SEQ ID NO: 421, and a common light chain comprising amino acid sequence SEQ ID NO:
 414. 50. A population of host cells in vitro, wherein each host cell comprises recombinant DNA encoding a bispecific antibody according to any of claims 1 to
 44. 51. A kit for production of a bispecific antibody according to any of claims 1 to 44, comprising an antibody heavy chain comprising a first VH domain as defined in any of claims 1 to 44, or nucleic acid encoding said heavy chain, an antibody heavy chain comprising a second VH domain as defined in any of claims 1 to 44, or nucleic acid encoding said heavy chain, an antibody light chain comprising a first VL domain as defined in any of claims 1 to 44, or nucleic acid encoding said light chain, and an antibody light chain comprising a second VL domain as defined in any of claims 1 to 44, or nucleic acid encoding said light chain.
 52. A kit according to claim 51, comprising a first heavy chain comprising amino acid sequence SEQ ID NO: 419 or SEQ ID NO: 426, or nucleic acid encoding said first heavy chain, a second heavy chain comprising amino acid sequence SEQ ID NO: 421, or nucleic acid encoding said second heavy chain, and a common light chain comprising amino acid sequence SEQ ID NO: 414, or nucleic acid encoding said common light chain.
 53. A method of producing a bispecific antibody according to any of claims 1 to 44, comprising culturing host cells according to claim 48 or claim 49 under conditions for expression of the bispecific antibody, and recovering the bispecific antibody from the host cell culture.
 54. A composition comprising a bispecific antibody according to any of claims 1 to 44, or isolated nucleic acid according to claim 47, in solution with a pharmaceutically acceptable excipient.
 55. A method of controlling bleeding in a haemophilia A patient, comprising administering a composition according to claim 54 to the patient.
 56. A composition according to claim 54 for use in a method of treatment of the human body by therapy.
 57. A composition according to claim 54 for use in a method of controlling bleeding in a haemophilia A patient.
 58. Use of a bispecific antibody according to any of claims 1 to 44 for the manufacture of a medicament for controlling bleeding in a haemophilia A patient.
 59. A method, composition for use, or use, according to any of claims 55 to 58, wherein the patient is resistant to treatment with FVIII owing to the presence of inhibitory antibodies in the bloodstream.
 60. A method, composition for use, or use, according to any of claims 55 to 59, wherein the patient is resistant to treatment with another bispecific antibody to FIXa and FX owing to the presence of inhibitory antibodies in the bloodstream.
 61. A method, composition for use, or use, according to claim 60, wherein the patient is resistant to treatment with emicizumab.
 62. A method of reducing development of inhibitory anti-drug antibodies in a haemophilia A patient undergoing treatment with a polypeptide that replaces FVIIIa activity, comprising administering a first FVIIIa-activity replacing polypeptide drug to the patient for a period of 1-12 months, switching the patient to a second, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, and switching the patient to either the first antigen-binding molecule or to a third, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, wherein the first, second or third FVIIIa-activity replacing polypeptide drug is a bispecific antibody according to any of claims 1 to 44, and wherein in each case the FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid is administered in a therapeutically effective amount to functionally replace FVIIIa in the patient, and wherein the risk of the patient developing inhibitory anti-drug antibodies to any of the FVIIIa-activity replacing polypeptide drug is reduced compared with a patient continuing to receive treatment with that FVIIIa-activity replacing polypeptide drug.
 63. A composition comprising a FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid, for use in a method of treating a haemophilia A patient while reducing develoment of inhibitory anti-drug antibodies, or use of a FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid for the manufacture of a medicament for use in a method of treating a haemophilia A patient while reducing development of inhibitory anti-drug antibodies, the method comprising administering a first FVIIIa-activity replacing polypeptide drug to the patient for a period of 1-12 months, switching the patient to a second, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, and switching the patient to either the first antigen-binding molecule or to a third, different FVIIIa-activity replacing polypeptide drug for a period of 1-12 months, wherein the first, second or third FVIIIa-activity replacing polypeptide drug is a bispecific antibody according to any of claims 1 to 44, and wherein in each case the FVIIIa-activity replacing polypeptide drug or its encoding nucleic acid is administered in a therapeutically effective amount to functionally replace FVIIIa in the patient, and wherein the risk of the patient developing inhibitory anti-drug antibodies to any of the FVIIIa-activity replacing polypeptide drug is reduced compared with a patient continuing to receive treatment with that FVIIIa-activity replacing polypeptide drug.
 64. A method according to claim 62, or a composition for use or use according to claim 63, wherein the first, second and third FVIIIa-activity replacing polypeptide drugs are recombinant or plasma-derived FVIII, emicizumab, and a bispecific antibody according to any of claims 1 to 44, in any order.
 65. A method, composition for use or use according to any of claims 55 to 64 wherein the treatment comprises subcutaneous administration of the composition to the patient. 